Research Spotlight

Rahimi, R. S. and J. G. O’Leary (2016). “Transfusing common sense instead of blood products into coagulation testing in patients with cirrhosis: Overtreatment not equal safety.” Hepatology 63(2): 368-370.

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In patients with cirrhosis the imbalance of procoagulants and anticoagulants combined with potential alterations in fibrinolysis and platelet number and function can alter standard laboratory coagulation testing. Prothrombin time and platelet count are frequently abnormal and, in our risk-averse health care system, often result in preprocedure transfusions to achieve “safer” thresholds. But what are we actually achieving: a risk of portal hypertensive bleeding, transfusion reaction, transfusion-related acute lung injury, infection transmission, human leukocyte antigen (HLA) antibody development, superior test results, or improved coagulation? According to the International Symposium on Coagulopathy and Liver Disease, rebalanced hemostasis is what we need to measure, which stems from both the prohemostatic and antihemostatic pathways working in concert. Disproportionately fewer nonportal hypertension-related bleeding complications occur in patients with cirrhosis relative to their prothrombin time and platelet values. This results from relatively rebalanced hemostasis. Despite this, many health care professionals continue to transfuse blood products (i.e., fresh frozen plasma [FFP] and platelets) prophylactically to improve laboratory profiles, regardless of function and prior data demonstrating a low risk of bleeding complications in patients with cirrhosis, especially for inimally invasive procedures like paracentesis. Fortunately, the study by De Pietri and colleagues in this issue of Hepatology offers an alternative to unnecessary preprocedural use of blood products in patients with cirrhosis with an elevated international normalized ratio (>1.8) and/or thrombocytopenia (<50 × 103/µL) using thromboelastography (TEG) to guide transfusions.4 TEG was adopted into clinical practice in 1985, more than 35 years after the procedure was first pioneered for research purposes. Using approximately 0.35 mL of whole blood in an oscillating cup at 37oC, this test uses mechanically transduced waves at different rates to graphically illustrate the patient's coagulation function. TEG is already used clinically in liver transplant, cardiovascular, trauma, and obstetric surgery where dynamic changes in blood volume and coagulation occur. This semiautomated bedside instrument easily yields an overall coagulation assessment in 30 minutes, with prolonged times resulting in blood product transfusions. A prolonged reaction time measures clot formation, indicative of coagulation factor function and results in transfusion (i.e., usually FFP), while a prolonged maximum amplitude measures clot strength, indicative of platelet function and results in transfusion. The authors of this open-label, intention-to-treat trial randomized patients to TEG-guided transfusion where patients only received transfusion if their functional coagulation was altered versus standard of care preprocedural prophylactic transfusion therapy.4 Of note, only 16.7% of TEG-guided patients received transfusions compared to 100% of patients in the standard of care arm (P = 0.009). Although platelet levels >50,000/µL have sufficient thrombin production, the use of FFP and platelet transfusions in patients with cirrhosis rarely, if ever, achieves complete normalization of coagulation parameters by standard testing. Of note, although allogenic transfusions at times are necessary, their cost and more importantly risk must also be taken into account (Table 1). The risk of overtransfusion was highlighted in a recent randomized controlled trial comparing liberal (hemoglobin <9 g/dL) versus restrictive (hemoglobin <7 g/dL) pack red blood cell (RBC) transfusion strategies in acute upper gastrointestinal bleeding. This trial subsequently resulted in a lower hemoglobin threshold (<7 g/dL) for RBC transfusion in general clinical care. Furthermore, liberal blood transfusions in patients with cirrhosis, compared to restrictive strategies, had the additional risk of worsening portal hypertension (P = 0.03) and resulted in higher bleeding complication rates (P = 0.01), a longer length of stay (P = 0.01), and more adverse events (P = 0.02; especially transfusion associated overload, P = 0.001), culminating in an increased all-cause 45-day mortality (P = 0.02).

Fernandez, H., J. Weber, K. Barnes, L. Wright and M. Levy (2016). “Financial Impact of Liver Sharing and Organ Procurement Organizations’ Experience With Share 35: Implications for National Broader Sharing.” Am J Transplant 16(1): 287-291.

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The Share 35 policy for organ allocation, which was adopted in June 2013, allocates livers regionally for candidates with Model for End-Stage Liver Disease scores of 35 or greater. The authors analyzed the costs resulting from the increased movement of allografts related to this new policy. Using a sample of nine organ procurement organizations, representing 17% of the US population and 19% of the deceased donors in 2013, data were obtained on import and export costs before Share 35 implementation (June 15, 2012, to June 14, 2013) and after Share 35 implementation (June 15, 2013, to June 14, 2014). Results showed that liver import rates increased 42%, with an increased cost of 51%, while export rates increased 112%, with an increased cost of 127%. When the costs of importing and exporting allografts were combined, the total change in costs for all nine organ procurement organizations was $11 011 321 after Share 35 implementation. Extrapolating these costs nationally resulted in an increased yearly cost of $68 820 756 by population or $55 056 605 by number of organ donors. Any alternative allocation proposal needs to account for the financial implications to the transplant infrastructure.

Arning, E. and T. Bottiglieri (2016). “Quantitation of S-Adenosylmethionine and S-Adenosylhomocysteine in Plasma Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.” Methods Mol Biol 1378: 255-262.

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We describe a simple stable isotope dilution method for accurate determination of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in plasma as a diagnostic test. SAM and SAH are key metabolic intermediates of methionine metabolism and the methylation cycle. Determination of SAM and SAH in plasma was performed by high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Calibrators (SAM and SAH) and internal standards ((2)H3-SAM and (2)H4-SAH) were included in each analytical run for calibration. Sample preparation involved combining 20 muL sample with 180 muL of internal standard solution consisting of heavy isotope labeled internal standards in mobile phase A and filtering by ultracentrifugation through a 10 kd MW cutoff membrane. Sample filtrate (3 muL) was injected by a Shimadzu Nexera LC System interfaced with a 5500 QTRAP((R)) (AB Sciex). Chromatographic separation was achieved on a 250 mm x 2.0 mm EA:faast column from Phenomenex. Samples were eluted at a flow rate of 0.20 mL/min with a binary gradient with a total run time of 10 min. The source operated in positive ion mode at an ion spray voltage of +5000 V. SAM and SAH resolved by a gradient to 100 % methanol with retention times of 6.0 and 5.7 min, respectively. The observed m/z values of the fragment ions were m/z 399 –> 250 for SAM, m/z 385 –> 136 for SAH, m/z 402 –> 250 for (2)H3-SAM, m/z 203 –> 46. The calibration curve was linear over the ranges of 12.5-5000 nmol/L for SAM and SAH.

Arning, E. and T. Bottiglieri (2016). “Quantitation of 5-Methyltetrahydrofolate in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.” Methods Mol Biol 1378: 175-182.

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We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5-MTHF) as a clinical diagnostic test. 5-MTHF is the main biologically active form of folic acid and is involved in regulation of homocysteine and DNA synthesis. Measurement of 5-MTHF in CSF provides diagnostic information regarding diseases affecting folate metabolism within the central nervous system, in particular inborn errors of folate metabolism. Determination of 5-MTHF in CSF (50 muL) was performed utilizing high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). 5-MTHF in CSF is determined by a 1:2 dilution with internal standard (5-MTHF-(13)C5) and injected directly onto the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve (25-400 nM) and has an analytical measurement range of 3-1000 nM.

Arning, E. and T. Bottiglieri (2016). “Quantification of gamma-Aminobutyric Acid in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.” Methods Mol Biol 1378: 109-118.

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We describe a simple stable isotope dilution method for accurate and precise measurement of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter in human cerebrospinal fluid (CSF) as a clinical diagnostic test. Determination of GABA in CSF (50 muL) was performed utilizing high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Analysis of free and total GABA requires two individual sample preparations and mass spectrometry analyses. Free GABA in CSF is determined by a 1:2 dilution with internal standard (GABA-D2) and injected directly onto the HPLC-ESI-MS/MS system. Determination of total GABA in CSF requires additional sample preparation in order to hydrolyze all the bound GABA in the sample to the free form. This requires hydrolyzing the sample by boiling in acidic conditions (hydrochloric acid) for 4 h. The sample is then further diluted 1:10 with a 90 % acetonitrile/0.1 % formic acid solution and injected into the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve and is linear from 6 nM to 1000 nM and 0.63 muM to 80 muM for free and total GABA, respectively.