Consultative Coagulation How to Effectively Answer Common Questions About Hemostasis Testing Session #5020

Consultative Coagulation

How to Effectively Answer Common Questions About Hemostasis Testing

Session #5020

Dorothy M. (Adcock) Funk, M.D.

Colorado Coagulation

Karen A. Moser, M.D.

Saint Louis University School of Medicine

October 9, 2014

Disclosures

Dr. (Adcock) Funk and Dr. Moser:

In the past 12 months, I have not had a significant financial interest or other relationship with the manufacturer(s) of the product(s) or provider(s) of the service(s) that will be

discussed in my presentation.

Topic Overview

• Direct Oral Anticoagulants

• Effects on coagulation assays

• Mixing Tests (Mixing Studies)

• Use and pitfalls

• Case examples will be provided during

both course sections

Brief Overview of Effects on Coagulation Assays

Direct Oral Anticoagulant

Agents

New Oral Anticoagulant Agents

• Direct Thrombin Inhibitors

• Dabigatran

• Trade Name: Pradaxa®

• Direct Xa Inhibitors

• Rivaroxaban

• Trade Name: Xarelto®

• Apixaban

• Trade Name: Eliquis®

• Edoxaban

• Not FDA approved as of yet

Pradaxa® is a registered trademark of Boehringer Ingelheim Pharma GmbH & Co.

Xarelto® is a registered trademark of Bayer Aktiengesellschaft.

Eliquis® is a registered trademark of Bristol-Myers Squibb Company.

FXI FIX FXII

FXa

FVII

FII Thrombin

Fibrinogen Fibrin FVIII

FV

 APTT

Intrinsic pathway

 PT

Extrinsic pathway

 TCT

Coagulation Cascade : In vitro model

DXa DTI

FIIa

 PT

 APTT

*Hawes E, Deal A, Jeanneret, et al. J Thromb Haemost. 2013;11:1493-1502.

Dabigatran Effect on APTT and PT

On therapy range Upper limit of normal range

Dabigatran – TT Response

0 50 100 150 200 250 300

0 50 75 100 150 200 300 400

Thr om bi n T im e ( s)

Dabigatran measured by LC-MS/MS ng/mL

*Hawes E, Deal A, Jeanneret, et al. J Thromb Haemost 2013;11:1493-1502.

Rivaroxaban Effect on APTT and PT

Francart S, Hawes E, Deal A, et al. Thromb Haemost 2014 doi 10.116-0/TH13-10-0871

On therapy range Upper limit normal range

Dabigatran and Rivaroxaban (Apixaban) Effect on Coagulation Assays

• Drugs act as inhibitors in the laboratory:

• Incomplete correction with 1:1 plasma mix

• Non specific inhibitor effect in factor assays

• Can cause a false positive Bethesda assay

• False positive Lupus Anticoagulant Assays

• Falsely elevated (normal) APCR, protein C clot-based activity and protein S activity and possibly

antithrombin activity (depending on drug and method)

• Dabigatran: Falsely low FXIII activity

• No effect on: D-dimer, VWF assays, free protein S

antigen, chromogenic protein C activity, reptilase time

Effect of Dabigatran on Factor Assays

0 25 50 75 100 125 150 200 300 400 500

FVIII 0.99 0.66 0.51 0.48 0.38 0.32 0.25 0.16 0.09 0.07 0.06 FIX 1.40 0.80 0.66 0.60 0.50 0.42 0.37 0.28 0.16 0.09 0.08 FXI 0.93 0.80 0.66 0.64 0.58 0.51 0.46 0.41 0.30 0.24 0.20

0 0.5 1 1.5

Factor Activity in IU/dL

* Adcock DM, et al. AJCP. 2013;139:102-1104.

Intrinsic Factors

“APTT-Based”

Extrinsic Factors

“PT-Based”

Dabigatran and Rivaroxaban Effect on Coagulation Assays

25 0 50 75 100 125 150 175 200

% c ha ng e c om pa re d to R ef er enc e Pl as ma

Laboratory Assays

Dabigatran Rivaroxaban

PC and PS assays: clot-based AT assays either IIa or Xa based

* Adcock DM, et al. AJCP. 2013;139:102-109; ECAT Proficiency Data 2012.

Case # 1

• 33 y/o woman suffers a post-partum DVT and is treated with rivaroxaban. Her physician orders a thrombophilia work-up; patient has the lab work drawn 2 days after starting tx.

• 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)

• dRVVT confirm is positive (ratio 1.3) with negative Staclot® LA and negative aCL and B2GP1 IgG and IgM

Staclot® is a registered trademark of Diagnostica Stago.

Case # 1

• The patient’s clinician calls you to discuss the results and you respond:

- PS activity may be falsely (elevated or decreased) on rivaroxaban?

- PC chromogenic activity may be falsely (elevated or decreased) on rivaroxaban?

- APCR ratio may be falsely (elevated or decreased) on rivaroxaban?

- LA assay results may be falsely (positive or negative) on

rivaroxaban?

Case # 2

• 82 year old man with atrial fibrillation is on

dabigatran and presents to the ED with bleeding and an elevated APTT and PT. He is in acute renal failure.

A FVIII activity with Bethesda titer is ordered:

- Factor VIII Activity: 8% (50-150%) - Bethesda Titer: 1.2 BU (<0.8 BU)

What do you tell the clinician?

Use and Pitfalls

Mixing Tests (Mixing Studies)

Mixing Tests- Overview

• What are plasma mixing tests (mixing studies)?

• How are they performed?

• In what situations are they useful?

• How are they interpreted?

• Practical examples

Plasma Mixing Test

• Used in the evaluation of a prolonged APTT (most commonly) and/or PT

• Screen for determining whether prolongation is due to a factor deficiency or inhibitor

• Can be performed with other assays

• e.g. DRVVT, VWF activity, FXIII activity

• Performance and interpretation varies

• Very few published guidelines or standards

The ART of coagulation testing!

FXI FIX FXII

FX

FVII

FII Thrombin

Fibrinogen Fibrin FVIII

FV

APTT

Intrinsic pathway

PT

Extrinsic pathway

TCT

Coagulation Cascade: In vitro model

Mixing Tests (APTT or PT)

Normal Plasma Mixing Test

50%

+ 1:1

Patient

↑ APTT or PT

Normal Pooled Plasma

Perform APTT or

PT on Mixture

Results

1. Corrects 2. Fails to

Correct

50%

**Principle- at least ~50%

factor is present in the

mixture, which is adequate

to correct APTT or PT in

cases of factor deficiency

Mixing Tests (APTT or PT)

Correction = Factor Deficiency

If immediate correction observed, rule out time/temperature dependent inhibitor

No/partial correction or prolongation

with incubation = Factor Inhibitor

Mixing Tests- Technical Variables

• Source of normal plasma

• Ratio of patient plasma to normal plasma

• 1:1 or 4:1 (for a weak inhibitor)

• Method to determine time/temperature dependence

• Criteria for correction

• Based on specific value versus formula

Source of Normal Plasma (NP)

• Pooled (n > 20) normal plasmas, fresh or frozen

• Do not use plasma from previously “normal” APTT or PT sample

• Lyophilized plasmas generally not recommended

• Platelet poor (< 5 -10X 10

/L)

• Well characterized

• Known factor activities (close to 100% of all factors)

• ↑ FVIII can cause false negative study

• Should be screened and LA negative

Incubated Mixing Test

50%

Patient NP Patient + NP

Separately incubated

patient + NP Mix (Control) Patient + NP

incubated together (Incubated Mix) Incubate each at

37º C for 60 to 120 minutes, then mix patient with NP

Compare Incubated Mix to Control

50%

What is Correction?

Mixing Test Results

Definitions of Correction

• No consensus or standard definition

• Correction in relation to reference interval

• Upper limit of 2 SD or 3 SD

• Upper limit + 5 seconds

• Correction in relation to normal pooled plasma (NP)

•

NP + 5 seconds

• NP plus 10%

• Correction in relationship to mean normal clotting time

• Mean normal clotting time + 2SD or +3SD

• Rosner index of <15*

• Percent correction*

• Other laboratory-defined criteria

* Index and % correction cutoff must be established by each laboratory

Definitions of Correction, cont.

• Correction in relation to reference range or normal pool

• Weak inhibitor with minimal prolongation of APTT or PT may correct into normal range with mixing

• Factor deficiency with prolonged PT may not correct to normal with mix

• May result from PIVKA interference

• Calculations (% correction or Rosner index)

• Cut-offs are reagent and instrument specific

• Each laboratory must determine own cut-offs

Published Calculations for Mixing Study Correction Determination

• Percent Correction

• Chang SH, Tillema V, Scherr D. Am J Clin Pathol. 2002; 117:62-73.

• Rosner Index

• Rosner E, Pauzner R, Lusky A, et al.

Thromb Haemost. 1987; 57(2):144-147.

Percent Correction

Am J Clin Pathol. 2002;117:62-73.

Percent Correction

PP APTT – 1:1 Mix APTT PP APTT – CNP APTT

X 100

=

PP – patient plasma; CNP – citrated normal plasma

APTT*

• >75% correction - Factor deficiency

• < 58% correction – Inhibitor PT*

• > 75% correction – Factor deficiency

• < 70% correction - Inhibitor

*Percent Correction cutoff should be verified by each laboratory

Rosner Index

Thromb Haemost. 1987; 57(2):144-147.

A = Clotting Time of Patient

B = Clotting Time 1:1 Mixing Test

C = Clotting Time of normal pooled plasma (NP)

High index (15 or greater) suggests inhibitor*

Low index suggests factor deficiency*

Index = B – C

A X 100

* Index cut-off must be established by each laboratory

What about minimal prolongation?

• 3-5 s prolonged clotting time

• Mixing tests are difficult to interpret

• Are mixing tests indicated?

• 4:1 APTT plasma mix may be useful

• Weak inhibitor may correct into the normal

range

Follow Up

Prolonged clotting time (PT and/or APTT)

Factor deficiency more likely Inhibitor more likely

Appropriate factor activity assays Lupus anticoagulant and/or specific factor inhibitor assays

Corrects Does Not Correct

Mixing test (PT and/or APTT, depending on initially prolonged clotting time)

Adapted from Semin Thromb Haemost. 2013; 39:283-290.

Correlation with all

available clinical and

laboratory information is

essential

Time to Practice!

Case 3

Test Result (s) Reference Interval

PT 12.0 11.9-14.1

APTT 57.2 23.4-36.4

APTT 1:1 mix 33.3 23.4-36.4

APTT 1:1 incubated mix

35.2 NA

APTT 1:1 incubated control

35.5 NA

TT 11.0 <20

What are the abnormalities?

What is your differential diagnosis?

What additional testing is indicated?

Case 4

• ^Test Specific factor inhibitor Result (s) Reference Interval

PT 12.6 11.9-14.1

APTT 82.2 23.4-36.4

APTT 1:1 mix 58.0 23.4-36.4

APTT 1:1 incubated mix 70.2 NA

APTT 1:1 incubated control

68.4 NA

TT 13.6 <20

What are the abnormalities?

What is your differential diagnosis?

What additional testing is indicated?

Case 5

Test Result (s) Reference Interval

APTT 51.1 23.4-36.4

APTT 1:1 mix 35.0 23.4-36.4

APTT 1:1 incubated mix 44.3 NA

APTT 1:1 incubated control

36.4 NA

TT 9.0 <20

What are the abnormalities?

What is your differential diagnosis?

What additional testing is indicated?

Case 6

Test Result (s) Reference Interval

PT 35.8 11.9-14.1

APTT 110.4 23.4-36.4

APTT 1:1 mix 45.2 23.4-36.4

APTT 1:1 incubated mix 54.6 NA

APTT 1:1 incubated control

50.4 NA

TT 13.5 <20

What are the abnormalities?

What is your differential diagnosis?

What additional testing is indicated?

Case 7

What are the abnormalities?

What is your differential diagnosis?

What additional testing is indicated?

Test Result Reference Interval

APTT 135.2 23.4-36.4

APTT 1:1 mix 48.2 23.4-36.4

TT >150 <20

APTT (heparin neutralized)

106.5 23.4-36.4

TT (heparin neutralized) >150 <20

Conclusion- Mixing Tests

• Mixing tests can be a useful screening tool to distinguish the presence of a factor

inhibitor from factor deficiency

• Must be performed and interpreted with caution

• Guidelines and standards are needed

References – Direct Oral Anticoagulants

• Adcock DM. Coagulation assays and anticoagulant monitoring. Hematology 2012.

American Society of Hematology Education Program Book: 460-465.

• Adcock DM, Gosselin B, Kitchen S, Dwyre D. The Effect of Dabigatran on Select Specialty Coagulation Assays. Am J Clin Pathol. 2013;139:102-109.

• Hawes EM, Deal AM, Adcock D, Gosselin R, Jeanneret C, Cook AM, Taylor JM, Whinna HC, Winkler AM, Moll S. Performance of coagulation tests in patients on therapeutic doses of dabigatran: a cross-sectional pharmacodynamics study based on peak and trough levels. J Thromb Haemost 2013;11:1493-1502.

• Francart S, Hawes E, Deal A, Adcock D, Gosselin R, Jeanneret C, Friedman K, Moll S. Performance of coagulation tests in patients on therapeutic doses of

rivaroxaban. A cross-sectional pharmacodynamic study based on peak and trough plasma levels. Thromb Haem.2014. In press.

• Gosselin R, Hawes E, Moll S, Adcock D. Performance of various laboratory assays in the measurement of dabigatran in patients receiving therapeutic doses. A

prospective study based on peak and trough levels. Am J Clin Pathol 2014;141:262-267.

References- Mixing Tests

• Chang S, Tillema V, Scherr D. A “percent correction” formula for evaluation of mixing studies. Am J Clin Pathol. 2002; 117:62-73.

• Clinical and Laboratory Standards Institute (CLSI). One stage prothrombin time (PT) test and activated partial thromboplastin time (APTT) test; approved guideline- second edition. CLSI document H47-A2. Wayne, PA: CLSI, 2008.

• Kershaw G, Orellena D. Mixing tests: diagnostic aides in the investigation of prolonged PT and APTT. Semin Thromb Haemost. 2013; 39:283-290.

• Krishnan J. Coagulation testing. In: Kottke-Marchant K, ed. An Algorithmic Approach to Hemostasis Testing. Northfield, IL: CAP Press; 2008:78-81.

• Ledford-Kraemer M. All mixed up about mixing studies. In: The Clotting Times: The Official Newsletter of CLOT-Ed, Inc. 2004; 3(4):1-11.

• Pengo V, Tripodi A, Reber G, et al. Update of the guidelines for lupus anticoagulant detection. J Thromb Haemost.

• Sahud MA. Laboratory diagnosis of inhibitors. Semin Thromb Haemst. 2000;

26(2):195-203.

• Wagenman BL, Townsend KT, Mathew P, Crookston KP. The laboratory approach to inherited and acquired coagulation factor deficiences. Clin Lab Med. 2009; 29:229-252.