Maintaining Hemostasis and Anticoagulation on ECMO October 15, 2018

Jun Teruya, MD, DSc, FCAP

Professor of Pathology & Immunology, Vice Chairman for Education Professor of Pediatrics Professor of Medicine Baylor College of Medicine Chief of Division of Transfusion Medicine & Coagulation Texas Children’s Hospital Conflict of interest in the past 12 months. Nothing is related to this presentation.

Research Support/P.I. No relevant conflicts of interest to declare

Employee No relevant conflicts of interest to declare

Consultant No relevant conflicts of interest to declare

Major Stockholder No relevant conflicts of interest to declare

Speakers Bureau No relevant conflicts of interest to declare

Honoraria No relevant conflicts of interest to declare

Scientific Advisory Board Stago, Novaheart, and Octapharma

Off label use: bivalirudin Circuit of VA ECMO

©2018 Jun Teruya Indication for ECMO (Extracorporeal Membrane Oxygenation) • Cardiac disease (VA ECMO or central ECMO) – Myocarditis, dilated cardiomyopathy, post-bypass myocardial failure, septic shock, etc • Respiratory disease (VV ECMO or VA ECMO) – Pneumonia, ARDS/ALI, status asthmaticus, sickle cell acute chest syndrome, air leak syndromes, interstitial lung disease, foreign body obstruction, mediastinal mass, pulmonary hemorrhage, congenital diaphragmatic hernia, meconium aspiration, pulmonary hypertension, etc • Extracorporeal cardiopulmonary resuscitation (ECPR or ECMO CPR) Hemostatic Complications During ECMO

• Bleeding – Oozing from catheter insertion sites, intracranial bleeding, thoracic bleeding, abdominal bleeding, hematuria • Clot formation – Ischemic stroke, clots in oxygenator, pump, or cannula, deep vein thrombosis Am J Respir Crit Care Med 2017;196:762-771 Bleeding and Thrombosis Events are Different among Hospitals Bleeding and Thrombosis Events are Different among Hospitals Bleeding and Thrombosis Events are Different among Hospitals Bleeding and Clotting on ECMO

• Retrospective chart review (from12/2005 to 12/2014) that identified 30 patients who died while receiving or within 24 hours of the discontinuation of ECMO, and also had an autopsy performed. • Thirty patients [age 0.46 years (0.08-3.41), males 16 (53.3%)] were identified and of these, 29 patients (97%) were noted to have hemorrhage in at least one organ system on autopsy. Thrombosis was identified in 17 (57%) of the patients. Possible Etiology of Bleeding

• Coagulation factor deficiency including fibrinogen and factor XIII • Thrombocytopenia/platelet dysfunction • Overdose of anticoagulant • Acquired von Willebrand syndrome (AVWS) • Hyperfibrinolysis • Anatomical cause • Others Etiology of Thrombosis (Virchow’s Triad )

Procoagulant surface of circuit

Anticoagulant

Turbulance Slow blood flow

ECMO Day 15

Purpuric lesions on her chest and her right hand started to appear. Doppler was negative. Ped Crit Care Med, 2014;15:e198-205 Coagulation Activation Markers: Age-Based Difference

Reference Range: TAT 2-4.2 µg/L F1.2 69-229 pmol/L Fibrinolysis Activation Markers: Age-Based Difference

Reference range: PAP 400-700 µg/L D-dimer <0.40 µg/mL (plasmin antiplasmin complex)

Incidence of Acquired Coagulation Disorders in the First 7 Days of ECMO 100 Factor XIII Level During ECMO 80

40 p <0.001 20 p <0.001

0 Normal Plasma… Day 1… Day 5…

Median IQR (1st, 3rd) Normal Plasma 83.6% 80.7, 85.1 Day 1 59.6% 46.7, 61.6 Day 5 50.9% 42.3, 57.1

Unpublished data Intravascular Hemolysis

• Monitored by plasma hemoglobin level. – Reference range <30 mg/dL (azide methemoglobin measured with a dual wavelength photometer) • Due to clots, pump, and/or position of cannula. • Due to high hemoglobin of 13 g/dL.*

*J Intensive Care Med. 2017 Jan High Hemoglobin Is an Independent Risk Factor for the Development of Hemolysis During Pediatric Extracorporeal Life Support. Free hemoglobin

• Decreases NO • Renal failure • GI dystonia and pain • Systemic and pulmonary hypertension

Thrombogenic!

Rother, R. P. et al. JAMA 2005;293:1653-1662. Blood. 2015;126:2338-2340 Free Hemoglobin Is Thrombogenic Known Evidence Free hemoglobin • Augments platelet adhesion and microthrombi formation on fibrin(ogen), extracellular matrix, and collagen at high shear stress. • The effect was blocked by anti-GPIbα or depletion of VWF. • Binds to A1 domain of VWF and increases platelet deposition to fibrin(ogen)-coated surface. • Binds to A2 domain of VWF thus competing with ADAMTS13 for cleavage • Interacts with vWF A1 domain, affecting GPIbα-vWF interaction. Possible role of recombinant haptoglobin?

Pediatric Critical Care Medicine 2018 • Prospective study by the Collaborative Pediatric Critical Care Research Network • N = 216 patients • Peak plasma free hemoglobin: none (<0.1 mg/dL ), mild (0.1-50 mg/dL), moderate (50-100 mg/dL), or severe (>100 mg/dL) • Conclusion: Hemolysis may contribute to renal failure (hazard ratio 1.04), but was not associated with mortality (hazard ratio 1.01). • Deficiency of this paper: no assay method mentioned; specimen collection site not mentioned; repeat assay not mentioned. Anticoagulation Heparin vs Bivalirudin Indication of Bivalirudin

• Heparin induced thrombocytopenia (FDA approved) • Unable to achieve and maintain anti-Xa despite heparin of 40 units/kg/hour (off-label) • Persistent increased fibrin deposition in ECMO or VAD despite achieving and maintaining appropriate anti-Xa levels. (off label) Heparin vs Bivalirudin Heparin Bivalirudin Action • Binding to antithrombin • Binding to circulating and heparin cofactor II and clot bound thrombin  anti-Xa and anti-IIa • Release TFPI

Half life 90 min 25 min Clearance Kidney Binding protein 18 plasma proteins Only thrombin including AT and HCII Bioavailability Poor Good Monitoring PTT and anti-Xa PTT, dTT, ECT Antidote Protamine None (Heparin rebound may happen) Monitoring of Bivalirudin

• PTT at the target of 60-80 seconds is commonly used. • However, because PTT correlates poorly with unfractionated heparin levels, PTT may not be the best test for monitoring of bivalirudin, either. Comparison between Anti-Xa vs. PTT and ACT in Newborns

300 ACT y = 25.262x + 206.25 R2 = 0.0157 250 r= 0.125

200

ACT (sec) ACT

150

100

PTT y = 72.925x + 66.117 2 50 R = 0.1328 r= 0.364

PTT (sec) PTT (sec)

0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 Heparin Level (units/mL) PTT (with PTT Hepzyme <40 sec) n=316 ACT

Khaja, W, Bilen, O, Lukner, R, Edwards, R, Teruya, J. Am J Clin Pathol 2010;134:950-954.

• Poor correlation between ECA and PTT and dTT and PTT. • Further studies are needed to best monitor bivalirudin effect. Monitoring Heparin for ECMO and VAD at TCH Tests Desired Comments Target / Range PT <16.0-17.0 sec To know underlying coagulable state PTT hepzyme <38.0 sec Fibrinogen >200 mg/dL PTT Depending on the device To monitor heparin effect

D-dimer To monitor fibrin formation and fibrinolysis in the circuit and general circulation Platelet count >100,000/mm3

Heparin level 0.2-0.5 units/mL To monitor heparin anticoagulant (Anti-FXa) activity Antithrombin >100-120% To maximize heparin efficacy

 Reset hemostasis.  Plasma hemoglobin >150 mg/dL.  Acquired von Willebrand syndrome (confirmed or suspected), which cannot be managed by Humate-P™ or Vonvendi™.  Elevated bilirubin level that heparin anti-Xa cannot be measured.  In the setting of TAMOF (thrombocytopenia- associated multiple organ failure), min 5 days, max 14 days, everyday. Monitor OFI (organ failure index) for efficacy.  FFP should be used as replacement fluid in order to prevent dilutional coagulopathy.

Etiologies of Bleeding and Thrombosis and Possible Management Etiologies Management • Plasma • Coagulation factor deficiency • Platelets • Thrombocytopenia • Cryoprecipitate Acquired VWS Bleeding VW factor Hyperfibrinolysis Antifibrinolytic FXIII deficiency FXIII concentrate ECMO Intravascular hemolysis = “Reset” hemostasis High fibrinogen, FVIII, VWF • Heparin Thrombosis • Antithrombin Under anticoagulant Circuit clots • Bivalirudin Lupus anticoagulant Plasma exchange TAMOF Replace Acknowledgement People who made a significant contribution • Shiu-Ki (Rocky) Hui, MD

• Vadim Kostousov, MD

• Karen Bruzdoski, MT (ASCP) Hospital therapy dogs

• Amir Navaei, MD

My therapy dogs Thank you. [email protected] Maintaining Hemostasis and Anticoagulation on Extracorporeal Circuits

October 15, 2018 Learning Objectives: • Describe the composition of clots formed in extracorporeal membrane oxygenation circuits • Interpret tests utilized for monitoring anticoagulation on extracorporeal circuits • Name 2 possible mechanisms for acquired von Willebrand syndrome for patients on left ventricular assist devices

www.aabb.org 2 Sample Chart Graphic

1st Qtr 2nd Qtr 3rd Qtr 4th Qtr

www.aabb.org 3 www.aabb.org 4 Acquired von Willebrand Syndrome Associated with LVAD Implants

Jing-fei Dong

BloodWorks Research Institute Hematology Division, Department of Medicine, University of Washington School of Medicine Changing Blood Flow in Disease States

1. Disease States: atherosclerosis-aortic/carotid stenosis, arterial aneurysm, and arteriovenous malformation 2. Procedure-induced: angioplasty (stenting), transcatheter aortic valve replacement (TAVR), central vein catheter, extracorporeal membrane oxygenation (ECMO), Changing Blood Flow in Disease States

These are mechanical pumps

that drive or help to LVAD drive blood circulation. http://www.thoratec.com

Aorta Heartmate II Impella 5.0 (2.5) Duration Life-long Months to years Days Diameter (mm) 15 - 20 ~ 15 3 Volume (L/min) 4.5-6 5-6 5.0 (2.5)

Speed 60-80/min 6,000-10,000 rpm 33,000 rpm Catheter axial pump axial Catheter http://www.abiomed.com/impella/impella-25 Why do we need them? Why do we need them?

How many heart bits one has in a life time? Why do we need them?

Life time heart beats 80 beats x 60 mins x 24 hrs x 365 days x 80 yrs = ~3.3 x 109 Why do we need them?

A normal heart

(Congestive) Heart Failure

Life time heart beats 80 beats x 60 mins x 24 hrs x 365 days x 80 yrs = ~3.3 x 109 How to Treat?

1. The goal is to replace a failing heart with a young and vibrant heart. 2. ~2,000 heart transplants/yr in the US 3. ~800,000 people have NYHA Class IV heart failure (end-stage heart failure) in the US 4. Early attempts: xenografts and artificial hearts 5. Left ventricular assist device (LVAD): pulsatile vs. continuous (flow) pumps and bridge-to-transplant vs. destination therapy 6. These pumps save patient’s life, but complications are common. Complications Associated with LVAD implants (axial continuous LVAD)

1. Mechanical malfunction 2. Infection 3. Hemostatic complications

 Thrombosis/thromboembolism (stroke, 0.064 EPPY)  pump thrombosis (0.03 EPPY)  myocardial infarction (?)  non-surgical bleeding (0.63 EPPY, 19-40%)

Nascimbene A, et al, Blood 2016;127:3133 Non-Surgical Bleeding

1. Presentation: epistaxis, bleeding of the mediastinum and thorax, intracranial hemorrhage and gastrointestinal bleeding 2. Incidence: ~20% GI and ~11% ICH 3. Cause: 1) medications (anti-platelet drugs and anti-coagulants) and 2) pump related (changes in blood flow). 4. Treatment: Serve cases require blood transfusion, surgery, and/or modification of drug regimen. 5. GI bleeding: upper and lower GI, often at sites of arteriovenous malformation - acquired von Willebrand syndrome

Nascimbene A, et al, Blood 2016;127:3133 von Willebrand Factor (VWF) in Hemostasis Structure of von Willebrand Factor

VWF domain structure 250 kDa VWF multimerization VWF Activity and Related Disease States

Type 2A: deficient VWF binding to platelets (loss-of-function) Type 2B: increased VWF affinity for platelets (gain-of-function) - VWF clearance from plasma

NP 1 2A 2A 2B 3 UL NP von Willebrand disease TTP

TTP: thrombotic thrombocytopenic purpura LVAD and Acquired von Willebrand Syndrome (AVWS)

Patients with AVWS  High VWF Antigen  Reversible loss of large VWF multimers  Low ristocetin cofactor activity  Reduced collagen binding  Caused by increased VWF cleavage by ADAMTS-13 Inconsistencies  AVWS in > 95% patients, but bleeding in ~20% N: plasma from normal subjects B: baseline before LVAD  AVWS also in patients with thrombosis 3: 3 months after LVAD E: clinical events  No evidence of increased VWF cleavage Loss-of-Function vs. Gain-of-Function VWF

A high shear stress blood flow generated by a continuous LVAD can induce an open conformation of VWF to facilitate cleavage by the metalloprotease ADAMTS13 and to activate VWF for binding and activating platelets.

LOF

GOF

Shear-induced conformation change of VWF Loss-of-Function vs. Gain-of-Function VWF

LOF

GOF Which one causes bleeding in patients with LVAD implants?

Shear-induced conformation change of VWF Quantifying VWF Cleavage by ADAMTS-13

A ratio of MVTGNPASDEIK (EQAPNLVY/M1606VTGNPASDEIK) to ILAGPAGDSNVVK (A3) by mass spectrometry

Normal Patients (n = 6) subjects (n=26) baseline 1 month 3 month at event R value 0.23±0.11 0.22±0.07 0.16±0.1 0.18±0.06 0.14±0.09

Zhou Y, et al. ASAIO J, 63:849-853

Persistent High Shear Stress Activates VWF Patients on LVAD Patients

NS: normal control BL: baseline Dis: at Discharge 3M: 3 months after LVAD EP: explanted

healthy subjects healthy Nascimbene A, et al, JHLT 2014;33:470

Whole blood tests of Nascimbene A, et al JHLT 2017; 36:477 unpublished data AVWS and LVAD-Induced Bleeding and Thrombosis

Quantifying the rate of VWF cleavage by ADAMTS-13 distinguishes the two mechanisms.

Distinguishing the two mechanisms is clinically important (e.g., in transfusion).

Nascimbene A, et al, JHLT 2014;33:470 and Nascimbene A, et al JHLT 2017; 36:477 Summary

1. VWF cleaved by ADAMTS-13 accounts for < 1% of total circulating VWF in normal subjects. The rate of VWF cleavage changes, but does not correlate with clinical bleeding and/or thrombosis in patients on LVAD. 2. Loss of large VWF multimers can be caused by either excessive cleavage by ADAMTS-13 (loss-of-function) or VWF activation high shear stress to bind platelets without increase in ADAMTS-13 cleavage (gain-of-function). 3. The cause of LVAD-induced hemostatic complications goes far beyond VWF. But VWF can serve as a key mediator that activates platelets and endothelial cells, leading to different types of hemostatic complications. Acknowledgment

Bloodworks GIT Tristan Hilton Cheng Zhu Katie Houck Maria Nawrot ISB Jing Yang Qian Tian Dr. Barbara Konkle’s Lab Shizhen Qin

THI and UT Angelo Nascimbene O.H. “Bud” Frazier Arteriovenous Malformation

Question: Is it preexisting made apparent by LVAD or structural vascular defects induced by LVAD? Mayo Clinic Answer for now: Evidence appears to support both.

Snehal R. Patel, et al. JACC-HF 2017;4:962 Quantitative Measurement of VWF Cleavage

Issues with VWF multimer gel immunoblots: 1) sensitive to experimental conditions 2) variable antibody binding affinity 3) very low throughput 4) lack of quantitation Shear profile and VWF reactivity

Lack of repeated exposures Uncle Zhang JN, et al. (2002) Thromb. Hemost.88:817 Force on VWF (AFM)

Force-induced cleavage of VWF from Relaxation of VWF after force to the normal and patients native equilibrium state

Free energy landscape of the multimeric Wu T, et al. (2010) Blood 115:370 VWF after shear exposure (100 dyn/cm2 for 3 min at 37oC (Wijeratne SS, et al. Phys. Rev. Lett. 2013; 110:108102) Flow rate, pump speed, and pressure VWF Adhesive Activity

1. Ristocetin cofactor activity measures the agglutination of platelets by VWF (through the A1 domain) in the presence of ristocetin. 2. Collagen-binding measures primarily VWF binding to collagen type III through the A3 domain. Shear stress and cleavage

Nishio K, et al. PNAS 2004;101:10578 Bernardo A, et al. Unpublished Maintaining Hemostasis and Anticoagulation on Extracorporeal Circuits

10/15/2018 Faculty Disclosures

The following faculty have no The following faculty have a relevant financial relationships to relevant financial relationship: disclose: – Jun Teruya MD, DSc – Nabiha Saifee MD, PhD Octapharma: Member of – Jing-fei Dong MD, PhD data monitoring committee Evaheart: Member of data monitoring committee Stago: Member of Advisotry committee

www.aabb.org 2 Learning Objectives

• Describe the composition of clots formed in extracorporeal membrane oxygenation circuits • Interpret tests utilized for monitoring anticoagulation on extracorporeal circuits • Name two possible mechanisms for acquired von Willebrand syndrome for patients on left ventricular assist devices

www.aabb.org 3 Thrombosis and Anticoagulation during Extracorporeal Life Support

Nabiha Huq Saifee, MD, PhD Slides adapted from Wayne L. Chandler, MD Seattle Children’s Hospital

| | Pediatric ECLS

wikipedia ECLS and Hemostatic Balance

Artificial Surface Turbulent/slow blood flow Platelet activation Bleeding Leukocyte activation

Need for anticoagulation Thrombosis Coag factor consumption Platelet Loss RBC Loss

Peek et al. ASAIO 1999;45:250 http://mikela71.blogg.se/2011/march/ecmo.html Managing Hemostasis during Extracorporeal Life Support

ECLS: Balancing hemostatic activation on artificial surface which promotes clot formation on the circuit/device, with control of bleeding from vascular access points.

Options for managing ECLS hemostasis: 1. Transfusion - Replacement of platelets and coagulation factors 2. Monitoring/adjustment of anticoagulant levels – heparin 3. Replacement of ECLS circuit - $5,000 - $20,000; associated with bleeding and thrombotic risk ECLS Circuit Analysis

Gross and microscopic analysis of thrombi in ECMO circuits after removal. H&E and immunohistochemistry analysis of clot structure: tissue factor, fibrin, Von Willebrand factor, platelets

Pump Axle Clot, 90% of Circuits Pump Clot Fragments Inflow Side Pump Axle Clot Structure

Degraded RBCs Immunofluorescence

Fibrin

VWF, WBCs

Green = fibrin, Red = VWF, Blue = cell nuclei Oxygenator Membrane Clotting – Oxygenator Occlusion

Patient septic (necrotizing pneumonia), Delta Pressure hypercoagulable (multiple venous 100 80 thrombosis). Membrane occluded with 60 adherent clot. Delta pressure 9 to 95. 40 20 0 0.00 2.00 4.00 6.00 8.00

Delta Pressure (mmHg) Pressure Delta Day on ECMO

Membrane Tubule

Degraded RBC Fragment

Green = fibrin, Red = VWF, Blue = cell nuclei Thrombi associated with antifibrinolytic therapy

Venous Connector Clot Corner clot Full thickness

Clotting on hard plastic, venous and arterial sides

New Strut Clot

Old Axle Clot Arterial Tubing Clots – Embolic Risk

Coagulation activation on surface leads to fibrin deposition (green), followed by attachment of RBCs, platelets, macrophages

Fibrin attached to tubing Circuit Analysis Findings

Pump axle clot fragments are trapped in oxygenator, Not associated with oxygenator failure

Patients with hypercoagulable state (venous thrombosis) may be at increased risk of oxygenator clot formation and failure.

Antifibrinolytic therapy associated with unusual, sometimes extensive circuit clot.

Tubing thrombi start with fibrin at tubing surface Followed by expansion with trapped RBC, WBC, platelets. Likely thrombin mediated, surface activation mechanism. Reduction focused on blocking surface activation (anti-XIIa?) Heparin Monitoring - Three Tests

Sample Sensitive to factors Test Method type other than heparin

ACT Clot-based with Whole Platelet, coag (activated clotting contact activation Blood factors, inhibitors time) via Factor XII PTT Clot-based with Coag factors, (partial Plasma contact activation inhibitors thromboplastin time) via Factor XII

Anti-Xa heparin Anti-thrombin, free Plasma Chromogenic activity hemoglobin, bilirubin ACT monitoring Cardiopulmonary Bypass

High dose heparin is used during CPB: 3-5 U/mL Heparin is neutralized at the end of CPB with protamine. ACT is able to detect change from 0 to 4 U/mL

CPB

Baseline ACT Monitoring during ECLS (in vivo samples)

ACT vs Heparin Low dose heparin is used during 450 ECLS: 0.2-0.4 U/mL

400 R² = 0.0053 350 In this dose range there is no

300 correlation between ACT and

250 heparin level

200 ACT should be 150

abandoned for heparin Activated Clotting Time (Sec) Time Clotting Activated 100 monitoring during ECLS

0 0 0.2 0.4 0.6 0.8 1 1.2 Heparin Activity (aXa U/mL) Correlation of anti-Xa, PTT, and ACT with Unfractionated Heparin (UFH) in vitro samples

Spiked pooled normal plasma samples with UFH • Anti-Xa Heparin activity correlates well with UFH level • PTT correlates well with UFH level • ACT does not correlate with UFH level up to 0.6 U/mL Correlation of anti-Xa, PTT, and ACT with Unfractionated Heparin (UFH) in vitro samples

Spiked pooled normal plasma samples with UFH • Anti-Xa Heparin activity correlates well with UFH level • PTT correlates well with UFH level • ACT does not correlate with UFH level up to 0.6 U/mL ACT sensitive to Coagulation Factor Levels and Platelet Count (in vitro samples)

ACT decreases with ACT decreases with increasing Coag factors increasing platelet count Effect of multiple parameters on ACT during ECLS (in vivo samples)

ACT vs Platelet R² = 0.0891 ACT vs Fibrinogen R² = 0.121 ACT vs PT R² = 0.2546 450 450 450 400 400 400 350 350 350 300 300 300 250 250 250 200 200 200 150 150 150 100 100 100 50 50 50 0 0

0 Activated Clotting Time (Sec) Time Clotting Activated 10 20 30 40 50 (Sec) Time Clotting Activated 0 500 1000 1500 0 200 400 (Sec) Time Clotting Activated PT (Sec) Platelet Count (K/uL) Fibrinogen (mg/dL)

ACT is insensitive to heparin & affected by too many other factors • Prolonged with decreasing coag factor levels • Prolonged with low platelet counts • Prolonged with low fibrinogen Benefits of reducing ACT at Seattle Children’s Hospital

Goal: Use anti-Xa for heparin monitoring, reduce ACT use 24 ACTs/day to 12 ACTs/day

Results: Reduced line draws from 158/run to 78/run 80/run * 40 ~ 3200 tests and line draws/yr Reduced heparin dose changes from 66/run to 17/run 49/run * 40 ~ 1960 heparin dose changes/yr Estimated drop in phlebotomy related transfusion 7 to 3.5/run 20 neonates*3.5 ~ 70 transfusions Anti-Factor Xa Heparin Activity Assay

• Heparin-Antithrombin complex inhibits Factor Xa • Residual factor Xa activity is proportional to the rate of absorbance change (OD/min) • Greater change in Measure rate of absorbance change at 405nm absorbance means less heparin Adapted from Bates and Weitz, Circulation, 2005. Anti-Factor Xa Heparin Activity Assay

• Heparin-Antithrombin complex inhibits Factor Xa • Residual factor Xa activity is proportional to the rate of absorbance change (OD/min) • Greater change in absorbance means less heparin Issues with anti-Xa heparin activity during ECLS

Interference in chromogenic assay due to colored substances.

A significant number of pediatric patients on ECLS can develop high levels of free plasma hemoglobin and bilirubin due to circuit associated hemolysis.

Review of samples from pediatric patients on ECLS • 66 of 250 (26%) had plasma hemoglobin ≥ 70 mg/dL • 38 of 234 (16%) had total bilirubin ≥ 16 mg/dL Issues with anti-Xa heparin activity during ECLS

Hemoglobin Bilirubin Anti-Xa IU/mL Anti-Xa IU/mL UFH 0.6 U/mL 0.6 0.6

0.5 0.5

0.4 0.4

0.3 0.3 UFH 0.3 U/mL

0.2 0.2

0.1 0.1 0 100 200 0 6 12 Free Hb (mg/dL) Bilirubin (mg/dL)

Kostousov, Teruya, Arch Pathol Lab Med 2014;138:1503 Texas Children’s Hospital Effect of hemolysis on anti-Xa heparin activity

• Hemolysis was associated with increased rate of absorbance change in the assay, independent on heparin concentration – it occurred even in the absence of heparin. Similar for high bilirubin. • On average the increased rate of absorbance change as a function of plasma hemoglobin was 3.47±0.58x10-4 (OD/min)/(mg/dL). Effect of hemolysis on anti-Xa heparin activity

Heparin activity versus Plasma Hgb in Patient samples

Measured heparin activity does not exceed 0.4U/mL with plasma Hgb >200mg/dL Correction for hemolysis/icterus interference anti-Xa

Interference of hemolysis/icterus is concentration dependent.

Rate correction for hemoglobin = measured rate (OD/min) – ((plasma hemoglobin (mg/dL) * 3.47x10-4 (OD/min)/(mg/dL))

Assay may be corrected using results from STAT plasma hemoglobin or bilirubin assay. Heparin Monitoring: PTT versus anti-Xa heparin activity

Stable heparin therapy Normal PT Single sample per patient All pediatric patients Protamine PTT - Baseline PTT on heparin

Protamine added to PTT can neutralize heparin, producing estimate of baseline PTT. Baseline PTT was prolonged in >50% of pediatric patients on heparin.

Baseline PTT estimated using protamine can be used to correct the heparinized PTT back to normal baseline

Heparinized PTT Corrected PTT = ------(Pat PrPTT/Mean PrPTT)CI Effect of Corrected PTT

Original PTT versus aXa Corrected PTT with CI=1.5 versus aXa

Khan J and Chandler WL. Manuscript in press. Summary on Heparin Anticoagulation Monitoring

• Anti-factor Xa is the most sensitive test for heparin monitoring

• Anti-factor Xa can be falsely low in presence of high plasma free Hgb and bilirubin

• PTT may also be discordant with anti-factor Xa heparin activity

• Both anti-factor Xa and PTT may be corrected to improve accuracy Thank you

Wayne Chandler, MD

Jenna Khan, MD, PhD Chris Burke, MD Dana Matthews, MD Larissa Yalon, BSN, RN David McMullan, MD Tom Brogan, MD

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