ISLH 2019 NASCOLA Interesting Case Presentations

Dorothy M Adcock, M.D.Chief Medical Officer Laboratory Corporation of AmericaMay 10, 2019

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• Laboratory Test Results (15Apr2018)• APTT 16.6 sec (Normal 22.9-30.2)• FVIII one-stage H >500% (Normal 57-163)• FVIII BETH (one-stage) Cancelled (Normal < 0.8 BU/mL)

• Analyst questioned results• 2 year old known Hemophilia A with a high titer inhibitor • Analyst commented that FVIII was non-parallel • Analyst questioned whether sample may be activated

Case Study

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• Laboratory Test Results (12Mar2018)• APTT H 51.0 sec (Normal 22.9-30.2)• APTT 1:1 NP H 51.4 sec• Thrombin Time 19.0 sec (Normal <23.1)• FVIII one-stage L <1.0 % (Normal 57-163)• FVIII BETH(one-stage) H 200.2 BU/mL (Normal <0.8 BU/mL)• EDTA “presence ruled out”

Patient History: Last Available Assessment March 2018

Clinician was called to obtain more history: the boy was recently started on a new FVIII replacement product Hemlibra® (Emicizumab)

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In vivo coagulation scheme

Tenase complex

Prothrombinase complex

Mann K, et al. Hamostaseologie.2009(1):7-16

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1. Humanized recombinantFIXa/FX bispecific antibody

2. FVIII mimic that placesfactor IXa and FX into spatially appropriate positions

3. Development requiredevaluation of 40,000 bi-specific combinations and2,400 prototype antibodies

Emicizumab – kxwh (HEMLIBRA®) aka ACE910

Graphics from Calatzis et al. (2018) poster presented at 62nd Annual Meeting of the Society of Thrombosis and Haemostasis, Vienna Austria

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Emicizumab - kxwh

• Administered subcutaneously (versus intravenous for most FVIII products)• Effective in the presence of FVIII inhibitors and does not promote development of FVIII inhibitors• Half-life 4 – 5 weeks (versus 8 – 12 hours for FVIII)• Granted “breakthrough therapy” designation FDA approval in 2017 for routine prophylaxis or to prevent bleeding in adult and pediatric hemophilia A patients with factor VIII inhibitors, in 2018 FDA approved for hemophilia A patients without inhibitors

• Significant reduction in annualized bleeding rates reported

Cannot be monitored using the aPTT and the one-stage FVIII activity assay

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Emicizumab-kxwh Effect on Standard Laboratory Assays

• After stopping therapy, residual plasma levels may persist for up to 6 months

• Laboratory monitoring is not required with routine prophylactic dosing

• aPTT and aPTT-based assays (one-stage FVIII activity) should not be performed as they provide misleading results

• Chromogenic FVIII (human) assay will only provide assessment of emicizumab-kxwh activity

• Chromogenic FVIII (bovine) assay can measure residual FVIII and FVIII inhibitors

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Effects on APTT Assay

Adamkewicz et al. (2017), Poster presentation at Hemostasis & Thrombosis Research Society, Scottsdale, Arizona

Emicizumab normalizes aPTTresponse independent of aPTT reagent used

30-83 µg/mL

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Effects on FVIII One-Stage Activity Assay

A. Adamkewicz et al. (2017), Poster presentation at Hemostasis & Thrombosis Research Society, Scottsdale, ArizonaB. Calatzis et al. (2018), Presented at 62nd Annual Meeting of the Society of Thrombosis and Haemostasis, Vienna Austria

A.

B.

Emicizumab results in overestimation of FVIII activity in the one-stage FVIII activity assay

30-83 µg/mL

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Effects on Other APTT Based Coagulation Assays

Calatzis et al. (2017), Presented at XXXth International Society for Laboratory Hematology Meeting , Honolulu, Hawaii

Emicizumab strongly interferes in the APTT based Protein C and S activity assays as well as the Activated Protein C ratio

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Effects on FVIII Chromogenic Activity Assay

Adamkewicz et al. (2017), Poster presentation at Hemostasis & Thrombosis Research Society, Scottsdale, Arizona

Human FIXa & FX

Hyphen Biomed

Bovine FIXa & FX

Siemens Healthcare

Emicizumab co-factor activity is measureable in chromogenic assays containing human FIXa and FX, but is not measurable in assays using bovine origin FIXa and FX

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Effects on Other Chromogenic based Assays

Calatzis et al. (2017), Presented at XXXth International Society for Laboratory Hematology Meeting , Honolulu, Hawaii

Emicizumab does not effect chromo-genic based ATIII, Protein C and Plasminogen activity assays

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Effects on Latex Immuno Based Coagulation Assays

Calatzis et al. (2017), Presented at XXXth International Society for Laboratory Hematology Meeting , Honolulu, Hawaii

Emicizumab does not interfere in 3 latex particle based immuno-assays, the Free Protein S, the vWF antigen and vWF activity assay

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Effects on Two Thrombin Activated Coagulation Assays

Calatzis et al. (2018), Presented at 62nd Annual Meeting of the Society of Thrombosis and Haemostasis, Vienna Austria

Thrombin Time Fibrinogen Activity (Clauss)

Emicizumab does not effect the Thrombin Time and the Fibrinogen Clauss assay

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Additional Test Results Using Chromogenic FVIII Assay

• Laboratory Test Results (17Apr2018)• APTT 16.6 sec (Normal 22.9-30.2)• FVIII one-stage H >500% (Normal 57-163)• FVIII chromogenic L <8% (Normal 60.4-168.2)• FVIII BETH (one-stage) Cancelled (Normal <0.8 BU/mL)• FVIII BETH (chromogenic) H 196.9 CBU/mL (<0.6 CBU/mL)

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• Avoid APTT- based coagulation assays (e.g. APTT- based one-stage factor activity and FVIII Bethesda Inhibitor assays)1,2,3

• Where possible, replace APTT- based, with chromogenic or immunoassay - based coagulation tests

• Use chromogenic (bovine FIXa and FX) FVIII Bethesda assay when measuring FVIII inhibitors in patients on Emicizumab1,2,3

• For measuring Emicizumab a chromogenic (human FIXa and FX)1,2 or dilute one-stage assay with Emicizumab calibrator and controls can be used4

Recommendations for Coagulation Laboratories

1HEMLIBRA FDA Prescribing Information, 11/2017; 2MASAC Interim Guidance on Acute Bleed Management and Use of Laboratory Assays; November 24, 2017; 3Collins et al. (2018) Haemophilia 24: pp 344-347; 4Calatzis et al. (2018) Presented at 62nd Annual Meeting of the Society of Thrombosis and Haemostasis, Vienna Austria

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