M1722 Hot Topics in Hemostasis - capannualmeeting.org · Objectives • Describe the laboratory...

M1722 Hot Topics in Hemostasis

Russell A. Higgins, MD, FCAP

Karen A. Moser, MD, FCAP

© 2018 College of American Pathologists. Materials are used with the permission of the faculty.

Objectives

• Describe the laboratory assays currently available to

measure DOAC effect in individual patients.

• Evaluate DOAC interferences in common and esoteric

hemostasis assays.

• Assess the performance of your factor assays in the

presence of new extended half-life FVIII and FIX

replacement products.

• Review FVIII and FIX replacement product package inserts

for information pertaining to laboratory performance of

factor assays.

2© 2018 College of American Pathologists. Materials are used with the permission of the faculty.

Hot Topics in Hemostasis

Opening/Introductions

Measuring direct oral anticoagulants (DOAC) in the clinical laboratory

Measuring extended half-life FVIII and FIX replacement products in the clinical laboratory

Summary and Closing

Agenda


Measuring Direct Oral Anticoagulants (DOAC)

Karen A. Moser, M.D.

Assistant Professor, Pathology

University of Utah [email protected]

Medical Director, Hemostasis and Thrombosis

ARUP Laboratories© 2018 College of American Pathologists. Materials are used with the permission of the faculty.

Direct Oral Anticoagulant Agents

• Approval timeline

2010

2011

2014

2015

2017

Direct thrombin inhibitor

Direct Xa inhibitors5© 2018 College of American Pathologists. Materials are used with the permission of the faculty.

Terminology Matters…

• What to call these drugs?

– New oral anticoagulants (NOAC)

– Novel oral anticoagulants (NOAC)

– Non-VKA oral anticoagulant (NOAC)

• At least one literature report where NOAC

interpreted as “no anticoagulation”

– Oral direct inhibitor (ODI)

– Specific oral direct anticoagulant (SODA)

– Target-specific oral anticoagulants (TSOAC)

– Direct oral anticoagulants (DOAC)

• Recommended by ISTH

Barnes GD, Ageno W, Ansell J, Kaatz S for the Subcommittee on the Control of Anticoagulation. J Thromb Haemost. 2015; 13:1154-1156.


Coagulation Cascade (simplified)

Contact Factors

(XII, PK,

HMWK) XI

IX

VIIIa

X

XIIa

XIa

IXa

Xa

Va

II IIa (thrombin)

FibrinogenFibrin monomer

VII

Tissue

Factor

Negatively

charged surface

VIIa

Direct thrombin inhibitors

Direct Xa inhibitors

Extrinsic PathwayIntrinsic Pathway

Common Pathway


Direct Oral Anticoagulants

• Benefits

– No laboratory monitoring required per FDA labeling

– Rapid onset and cessation of anticoagulant effect

– Less risk of dietary interactions than warfarin

– May have less risk of bleeding complications than

warfarin

Shantsila E, Lip GYH. Non-Vitamin K Antagonist Oral Anticoagulants: A Concise Guide. New York: Springer: 2016.

Baglin T. Br J Haematol. 2014; 163(2):160-167.

Makam RCP, Hoaglin DC, McManus DD, et al. PLoS One. 2018; 13(5):e0197583. doi: 10.1371/journal.pone.0197583


Direct Oral Anticoagulants

• Challenges

– No laboratory monitoring required per FDA labeling

• Laboratory testing for these agents developing after

they are already on market

– Rapid onset and cessation of anticoagulant effect

• Less cushion for missed doses as with warfarin

– Twice daily dosing for dabigatranShantsila E, Lip GYH. Non-Vitamin K Antagonist Oral Anticoagulants: A Concise Guide. New York: Springer: 2016.

Baglin T. Br J Haematol. 2014; 163(2):160-167.

Tripodi A. J Thromb Haemost. 2016; 14(7):1325-1327.


Case 1

• A 57 year old man presents to the emergency department of

your hospital with symptoms concerning for an acute

stroke. He is unresponsive, but his wife tells the ED team

that he takes dabigatran for atrial fibrillation. His last dose

was this morning.


Case 1

• The ED physician calls the laboratory to ask how to tell

whether the patient has dabigatran on board prior to

initiating thrombolysis. You do not have a specific test for

measuring dabigatran validated in your laboratory.

What is your advice to your colleague?


When might we want to measure DOACs?

Emergent Results Needed

• Patients with acute

bleeding or thrombosis

on DOAC

• Overdose

• Trauma or emergent

operation needed in

patient on DOACTripodi A. Blood. 2013; 121(20):4032-4035. Tripodi A, Ageno W, Ciaccio M, et al. Blood Transfus. 2017; 13:1-9.

Douxfils J, Gosselin RC. Semin Thromb Hemost. 2017; 43:277-290. Baglin T, Hillarp A, Tripodi A, et al. J Thromb Haemost. 2013; 11:756-760.

Routine TAT Acceptable

• Impaired renal function (adjusted

dosing recommended for dabigatran,

rivaroxaban, edoxaban)

• Extremes of body weight

• Elective operative procedure in

patient on DOAC

• Suspected interactions with

concomitant drug

• Patients also receiving dual anti-

platelet therapy

• Assessment of compliance


Time Matters…

• Choice of assay in emergent versus routine clinical

situations will differ

13

Routine

• Favor quantitative assays

‒ More accurate

‒ Gives measurement of

concentration

‒ Usually requires

sendout to reference

laboratory

Emergent

• Favor qualitative assays*

‒ Routine clotting times

‒ Available in most

hospital laboratories

‒ Less accurate

‒ Variability between

reagents

* Unless quantitative assays validated locally. Use clotting times with caution, as shown in subsequent slides.

Douxfils J, Ageno W, Samama C-M, et al. J Thromb Haemost. 2018; 16:209-19.


Qualitative Assays- Options• APTT

– Relatively sensitive to dabigatran

• Normal APTT does not exclude presence of on-therapy levels of

dabigatran

– Not very sensitive to direct Xa inhibitors

• Insensitive to apixaban

14

Significant variability

between reagents

Baglin T, Hillarp A, Tripodi A, et al. J Thromb Haemost. 2013; 11:756-60.


Gosselin RC, Adcock DM, Bates SM, et al. Thromb Haemost. 2018; 118(3):437-450.

Kitchen S, Gray E, Mackie I, et al. Br J Haemotol. 2014; 166:830-841.


Qualitative Assays- Options• PT

– Not very sensitive to dabigatran

– Somewhat more sensitive to direct Xa inhibitors

• Rivaroxaban > edoxaban > apixaban

– Insufficiently sensitive, even if paired with normal APTT, to

completely exclude presence of DOAC

15

Significant variability

between reagents






Qualitative Assays- Options

• TT (thrombin time)

– Incredibly sensitive to dabigatran

• Normal TT excludes the presence of any level of dabigatran

– Not sensitive to direct Xa inhibitor

Note: There are currently limited data regarding performance

of betrixaban in hemostasis assays.

16





0

50

100

150

200

250

300

0 50 75 100 150 200 300 400

Thro

mb

in T

ime

(s)

Dabigatran measured by LC-MS/MS ng/mLHawes E, Deal A, Jeanneret, et al. J Thromb Haemost

2013;11:1493-1502.


Qualitative Assays- Options

• Anti-Xa activity (calibrated for UFH or LMWH)

– Use with caution!

• However, this may be a reasonable option in an emergent

situation with limited tests available

– Preferred to calibrate assay for direct Xa inhibitor you are

trying to measure

– At least one study suggests that anti-Xa assays calibrated

for UFH and LMWH could detect significant levels of DOAC1

• Others suggest that response may vary by kit2

17

1. Gosselin R, Grant RP, Adcock DM. Int J Lab Hematol. 2016; 38:505-513. 2. Sabor L, Raphael M, Dogne JM, et al. Thromb Res. 2017; 156:36-38.


Potential qualitative use of anti-Xa activity assays

18

Key-

Apixaban (diamond)

Edoxaban (triangle)

Rivaroxaban (circle)

Gosselin R, Grant RP, Adcock DM. Int J Lab Hematol. 2016; 38:505-513.


Quantitative Assays- Options for Dabigatran• Dilute thrombin time

– Dilute refers to patient plasma diluted in buffer and mixed

with NPP

– Linear increase directly proportional to increase in

dabigatran concentration

– Commercially available, not FDA approved

– Good accuracy reported between 50-500 ng/mL

• Adaptations to method may decrease LLOD

19






Quantitative Assays- Options for Dabigatran

• Ecarin-based methods

− Clotting time (ECT) or chromogenic assay (ECA)

− One commercial ECA kit available, not FDA approved

− LLOD ~14-46 ng/mL

− Not sensitive to heparins

• Chromogenic anti-factor II assay

− One commercial kit available, not FDA approved

− LLOD ~15 ng/mL

20

Prothrombin MeizothrombinEcarin

Inhibited by DTI

Not inhibited by heparin






Quantitative Assays- Options for Direct Xa inhibitors

• Anti-Xa chromogenic assays with DXa inhibitor-specific

calibrators

– Measures wide range of concentration, results ng/mL

• LLOQ ~30 ng/mL (varies by kit and drug)

• Some kits may require adaptation to measure 30-50 ng/mL

– No FDA approved calibrator materials

– Can also be elevated by heparin

• Complicates monitoring in patients transitioning to DXa inhibitor

21






Quantitative Assays- Option for all DOAC

• LC/MS-MS

– Considered gold standard for quantification

– Reportable range 5-500 ng/mL

– Only available in small number of reference laboratories

– No international reference standard available

– Timing of measurement matters

• Prefer peak or trough instead of random

22






Case 1, revisited

• What is your advice to your colleague?

– Thrombin time (TT) can be used qualitatively to assess

presence/absence of dabigatran.

– If TT is not available, APTT may be useful qualitatively, but

responsiveness depends on your local reagents.

– TT must be tested within 4 hours of sample collection (not a

problem in this emergent situation!)


Case 2

• A 30 year old woman experiences a DVT and is treated with

rivaroxaban. Her physician orders a thrombophilia work-up;

the patient has the lab work drawn 2 days after starting

therapy.

• The following assays were performed:

– Protein S activity (clot based): 65% (63 – 140%)

– Protein C activity (chromogenic): 72% (55 – 140%)

– APCR ratio: 2.2 (2.2 – 4.0)

– Positive dRVVT confirm (ratio 1.3)

– Negative APTT-based LA confirmatory assay

– Negative anticardiolipin and β2GP1 IgG and IgM

• How do you help her physician interpret these results?


DOAC Interferences- Selected AssaysAssay Dabigatran DXa inhibitors*

APCR ratio False Increase False Increase

AT activity - (Xa-based)

False Increase (IIa-based)

False Increase (Xa-based)

- (IIa-based)

PC activity- clot based False Increase False Increase

PC activity- chromogenic - -

PS activity False Increase False Increase

Free PS antigen - -

LA assays False Positive False Positive

APTT-based factor assays

(VIII, IX, XI, XII)

False Decrease False Decrease

PT-based factor assays (II, V,

VII, X)

False Decrease False Decrease

25

Mani H. Int J Lab Hematol. 2014; 36:261-268. Gosselin RC, Adcock DM, Bates SM, et al. Thromb Haemost. 2018; 118(3):437-450.

* Limited data on betrixaban


Case 2- revisited

• Results Interpretation in Presence of Rivaroxaban

– PS activity may be falsely increased.

• Risk of misclassifying PS deficiency as normal

– PC chromogenic activity is not affected.

– APCR ratio may be falsely increased.

• Risk of misclassifying APCR as normal

– LA assay results may be falsely positive.

– Anticardiolipin and β2GP1 IgG and IgM are not affected.


First reversal agent for dabigatran FDA approved October, 2015

Praxbind (idarucizumab)

Humanized Fab

Binds dabigatran to neutralize effect

First reversal agent for rivaroxaban and apixaban FDA approved May, 2018

AndexXa (andexanet alfa)

Recombinant FXa decoy molecule (inactive enzyme)

Binds DXa inhibitor in place of Fxa

Additional agent in development

Ciraparantag

(phase 2 study- reversal of apixaban and rivaroxaban)

Reversal Options

Almegren M. Vasc Health Risk Manag. 2017; 13:287-292. clinicaltrials.gov, ANNEXA-4 (identifier NCT02329327)

Greinacher A, Thiele T, Selleng K. Thromb Haemost. 2015; 113(5):931-942. cinicaltrials.gov, ciraprantag (identifier NCT 03288454, NCT03272910)

https://www.fda.gov/downloads/BiologicsBloodVaccines/CellularGeneTherapyProducts/ApprovedProducts/UCM606693.pdf


Summary

• While routine monitoring of DOAC is not required, several

clinical situations call for measuring DOAC in the

laboratory

• Choice of assay for measuring DOAC depends on clinical

question and how emergently the result is needed

• DOAC may interfere in a variety of routine and esoteric

coagulation assays, causing false negative or positive

results.


Monitoring New Factor VIII and IX Replacement Products

Russell Higgins, MD

Clinical Professor

Department of Pathology and Laboratory Services

University of Texas Health Science Center San Antonio

Medical Director, University Hospital System Pathology Services© 2018 College of American Pathologists. Materials are used with the permission of the faculty.

Factor VIII Variability

• Comparison of an old versus

new factor VIII method

• Main Variables

– Calibrator

– Reagent

– Instrument (mechanical

versus optical)

– Factor VIII deficient plasma-50.00

-40.00

-30.00

-20.00

-10.00

0.00

10.00

20.00

30.00

40.00

50.00

-10.0 10.0 30.0 50.0 70.0 90.0 110.0 130.0 150.0

Dif

fere

nce

(AMAX + TOP1)/2

Factor VIII:C Bias Plot


2017 CAP Survey (CGS3-02):

Factor VIII Activity (ISTH Assigned Value = 88 IU/dL)

CAP 2012 CAP 2013 CAP 2014 CAP 2015 CAP 2016 CAP 2017

Factor VIII Activity Reagent 1 -9 -9 -9 3 1 3

Factor VIII Activity Reagent 2 5 11 11 23 -1 15

Factor VIII Activity Reagent 3 -2 2 2 15 9 8

ISTH Assignment (IU/dL) 0 0 0 0 0 0

-25

-20

-15

-10

-5

0

5

10

15

20

25

% D

iffe

nce

(C

AP

-IS

TH)/

ISTH

BIAS -- ISTH Lot 4 Versus CAP


Native Factor VIII

• Some Variability

• Interlaboratory CV%

• ~12 (CAP CGE)

Recombinant Factor VIII Concentrates

• Some Variability


• 10, 10.9, 11.4 (Advate; 3 studies)

Modified Recombinant Factor VIII

Concentrates

• High Variability


• 16 (Eloctate)

• 14.7 (Adynovate)

• 122 (Bay 94-9027)

• High reagent and instrument dependent recovery

The Problem: variability of one-stage factor assays for

measuring modified factor products


Modified factor VIII replacement products

• Goal = Decrease immunogenicity or extend half-life

– Fusion proteins (e.g. albumin or Fc portion of IgG1)

– Pegylated factor VIII

– Single chain products (heavy and light chains covalently linked)


Factor VIII Assays

• Chromogenic (2 stages)

– Diluted patient plasma (FVIII

source) incubated with reagent

to activate Xa (Stage 1)

– Xa cleaves chromogenic

substrate (Stage 2)

– Not common in US laboratories

• One-stage factor assay

– Diluted patient plasma (FVIII

source) mixed with factor

deficient plasma

– aPTT-based(clot based)

– Most common factor assay


Modified Coagulation Factor Products

• Some extended half-life (EHL) recombinant products

*Graf L. Transfus Med Hemother 2018;45:86–91.

**Kitchen S, Tiefenbacher S, Gosselin R. Semin Thromb Hemost 2017;43:331–337.

Factor VIII Product Modification Half-Life (hours)

*Eloctate®; rFVIIIFc Fc fusion BDD rFVIII 19

*Adynovate®; BAX855 Pegylated rFVIII 14-16

*Afstyla®; rFVIII-SingleChain Single chain BD truncated rFVIII 14.5

**N8 GP (late stage) glycoPEGylated rFVIII 19

**Bay 94 9027 (late stage) PEGylated BDD rFVIII 18.2

Factor IX Product Modification Half-Life (hours)

*Alprolix®; rFIXFc Fc fusion protein 82

*Idelvion®, rFIX-FP Albumin fusion 102

*Rebinyn®; N9 GP Glycopegylated rFIX 93

Normal

FVIII

Half-Life

12 hours

Normal

FVIX

Half-Life

18-24

hours


What difference from labeled potency is

acceptable?

• No definition of acceptability

• Typically ~25% to 30% is acceptable

• Eloctate®

– One-stage and chromogenic

assays found to be acceptable

– Reflected in package insert

Sommer JM, Moore N, Mcguffie-Valentine B, et al. Haemophilia (2014), 20, 294–

300.

Eloctate


You receive a call from a Hematologist

“Can your the laboratory monitor Afstyla?”

A) Yes

B) No

C) Let me investigate and I will call you back


Afstyla® Field Study

• *one-stage factor VIII methods

underestimate Afstyla® by ~50%

• FDA approved with a conversion

factor of x2 across all one-stage

reagents

*Ledger KST, Feussner A, Kalina U, et al. Journal of

Thrombosis and Haemostasis, 16: 555–564

Blue bars are Afstyla

Red bars are Advate


N8-GP: recovery at two sites

• Most one-stage methods adequate

– APTT SP underestimates N8 GP

• “Some” overestimation by chromogenic

methods

Hillarp A, Bowyer A, Ezban M. Haemophilia (2017), 23, 458--465


BAY 94-9027

• PTT-A and APTT-SP

underestimate spiked samples

(right)

• Chromogenic methods had

adequate performance

– FVIII Chromogenic (Siemens)

– Electrochrome (IL)

– Coamatic (IL)

Church N, Leong L, Katterle Y. Haemophilia. 2018;1–10.


Modified Factor FVIII Products: Monitoring

*Graf L. Transfus Med Hemother 2018;45:86–91.

**Kitchen S, Tiefenbacher S, Gosselin R. Semin Thromb Hemost 2017;43:331–337.

Factor VIII Product Modification Half-Life (hours)

Monitoring Method

*Eloctate®; rFVIIIFc Fc fusion BDD rFVIII 19 Most one-stage or chromogenic

*Adynovate®; BAX855 Pegylated rFVIII 14-16 Most one-stage or chromogenic

*Afstyla®; rFVIII-SingleChain

Single chain BD truncated rFVIII 14.5 Most one-stage x 2 or chromogenic

**N8 GP (late stage) glycoPEGylated rFVIII 19 Most one-stage (avoid APTT-SP; SynthasILon Siemens Instrument) or chromogenic

**Bay 94 9027 (late stage) PEGylated BDD rFVIII 18.2 Most one-stage (avoid APTT-SP and APTT-A) or chromogenic


Modified FIX: A Bigger Problem


Rebinyn® (rFIX-GP): one-stage FIX recovery at two

sites

• Overestimated by

Pathromtin SL and APTT-SP

• Underestimated by Actin,

Actin FS, Actin FSL, and

SynthaSil

Bowyer AE, Hillarp A, Ezban M, et al. Journal of

Thrombosis and Haemostasis, 14: 1428–1435

!!! !!!


Alprolix® -rFIXFc

• Kaolin method underestimates

Alprolix®

• Silica-based reagent moderately

underestimates Alprolix®

– Manufacturer uses silica-based

reagent

– Authors concluded silica-based

reagent was okay Sommer JM, Buyue Y, Bardan S, et al. Thrombosis and Haemostasis

112.5/2014


Modified Factor FVIX Products: Monitoring

Kitchen S, Tiefenbacher S, Gosselin R.

Semin Thromb Hemost 2017;43:331–337.

Rebinyn®

Alprolix® Idelvion®


Potency Labeling: methodology

• European Pharmacopoeia

– FVIII labeled with chromogenic

– FIX labeled with one-stage

• Other Countries

– For US, either chromogenic or

one-stage

• *SSC/ISTH Recommendations

*Dodt J, Hubbard AR, Wicks J, et al. Haemophilia (2015), 21, 543--549


Which WHO?

• In the laboratory,

reference plasmas are

traceable to WHO IS for

Plasma rather than

Concentrate

• Differences exist btw

standards by design*

* Lollar P. Journal of Thrombosis and Haemostasis 2003;1:2275-2279.

**Thromb Haemost. 2003 Dec;90(6):1088-93.

***Sommer JM, Buyue Y, Bardan S, et al. Thrombosis and Haemostasis 112.5/2014

Post-infusion samples measured by clinical laboratories against WHO IS

for factor IX in Plasma

Product labeled in IU by appropriate test

Manufacturer’s internal reference material

WHO International Standard for Concentrate

***10%-20% Difference with

BeneFIX

**~+20%

overestimation of

Refacto Laboratory

Standard

discovered, 2003


Solutions?

• Product specific calibrators

• Additional method (chromogenic assay or additional one-stage)

• Correction factors when appropriate

• Send to reference laboratory when product cannot be measured


Solutions: product

specific calibration

• European Experience

– Refacto AF® Laboratory Standard

• *BAX 855

– Product specific calibrator improved

recovery and precision across 11

laboratories (right)

• Lab must know what product is infused

• Difficult to manage multiple standards

*Bulla O, Poncet A, Alberio L, et al. Haemophilia (2017), 23, e335–e339


Solutions: Use chromogenic methodology

• Chromogenic assays have ?less variability?

• Diminished sensitivity at low factor levels

• No FDA approved chromogenic kits in US

• Chromogenic assays may not be expensive

– Aliquoting reagent reduces cost

**Kitchen S, Blakemore J, Friedman Kd, et al. Journal of Thrombosis and

Haemostasis, 14: 757–764

*Sommer JM, Moore N, Mcguffie-Valentine B, et al.

Haemophilia (2014), 20, 294–300.

*

**


Solutions: correction factors

• Blanket correction of one-stage

methods (x2) approved for

Afstyla®

– Some concern [e.g. Synthafax

or Actin® FS (right)]

*Ledger KST, Feussner A, Kalina U, et al. Journal of Thrombosis and Haemostasis, 16: 555–564

**Bowyer A, Key N, Dalton D, et al. DOI: 10.1111/hae.13290


Solutions: send to designated reference laboratory

• Long turn around time not appropriate for emergencies

• May be adequate for PK studies


Real World Solutions: Summary

• Modified coagulation factors are not recovered by all one-stage assays

• Use product inserts and literature to identify gaps in laboratory testing

• Develop relationship with treating physicians and pharmacists

• Communicate gaps in laboratory testing for products that cannot be

measured accurately

• Develop a local strategy to cover the gaps


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M1722 Hot Topics in Hemostasis - capannualmeeting.org · Objectives • Describe the laboratory...

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