Improving Acute Care

with Coagulation
Mixing Studies

George A Fritsma MS, MLS

The Fritsma Factor,
Your interactive Hemostasis Resource
Sponsored by Precision BioLogic
Dartmouth, Nova Scotia
[email protected]
www.fritsmafactor.com

1
 Coagulation Mixing
Studies
Learning Objectives
At the conclusion of this webinar, the participant
1. Prepares a stepwise PTT and PT mixing study protocol
2. Indicates the clinical purposes for PTT mixing studies
3. Explains why the mixing study is an acute care assay
4. Correlates mixing study results with coagulation test results

2
 Mixing Study
A First-line Investigation to
differentiate a
coagulation deficiency from an inhibitor
+ =

Kershaw GK, Orellana D. Mixing tests: diagnostic aides in the investigation of prolonged prothrombin
times and activated partial thromboplastin times. Semin Thrombos Hemost 2013;39:28390. 3
 Case: 32-yo Female
Pre-op Screen
Six weeks post-partum
Easy bruising, frequent nosebleeds,
menorrhagia

4
 Pre-op Screen
32-yo Female, 6 Weeks Post-partum

Assay Patient RI
HGB 11.8 g/dL 1215 g/dL
PT 12.4 s 9.812.6 s
PTT (APTT) 42.5 s 2535 s
PLT count 310,000/L 250450,000/L
Fibrinogen 320 mg/dL 220498 mg/dL
Isolated, prolonged PTT response? 1:1 PTT mix

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 Rule Out Heparin, Dabigatran
Assay Patient RI
TT 14 s <21 s

R/O dabigatran and unfractionated heparin (UFH)

Outpatientconsider dabigatran
Inpatientunrecorded UFH flush of vascular catheter
If dabigatran, discontinue, cancel order
If UFH, use Hepsorb (polybrene) or Hepzyme, proceed
If no UFH, perform 1:1 PTT mix to differentiate factor
deficiency from factor-specific inhibitor or non-specific
inhibitor lupus anticoagulant (LA)
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 PTT Mixing Study: Cheap and Basic
Start within 2 h to avoid specimen degradation
Factors V (FV) and VIII (FVIII) are labile
Platelet factors (mostly FV) released to plasma
Ensure pt plasma is platelet-poor (free), <10,000/uL
Mix plasma 1:1 with pooled normal plasma (NP) and perform
immediate PTT on mixture
PTT of 1:1 mix corrects to 10% longer than NP PTT
Factor deficiency
No correction: 1:1 mix is >10% longer than NP PTT
Non-specific inhibitor, usually LA
Specific inhibitor (anti-FVIII) may be present, usually requires 37C
incubation
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 PTT Mixing Study

+ =
Equal
volumes 1:1 mix
Patient Normal
plasma plasma

PTT PTT Manufacturers value

42.5 s 30 s confirmed by QA staff
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 100 uL 100 uL
PTT reagent CaCl2

+ = 1:1 mix +
NP value 1:1 mix + PTT rgt +
is 30s 100 uL PTT rgt CaCl2
1:1 mix

PTT Mixing Study PTT

Using 10% Rule 33 s: Correction
>33 s: No correction
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 1:1 PTT Mix with Incubation
PTT of immediate mix 10% longer than NP
Correction: factor deficiency? But first
Incubate 1:1 mix, 37C, 12 h and repeat
Correction after 37C mix = factor deficiency
Incubated PTT remains >10% longer than NP
Specific inhibitor such as anti-FVIII
IgG4: Temp dependent, may require incubation
However, some FVIII neutralization within 10 m
May detect in immediate mix

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1:1 PTT Mix After 37C Incubation

Only when unincubated mix corrects

Must also incubate normal control plasma
Compare mix PTT to incubated NP PTT
May also detect temp-dependent LA
~15% of LAs are temp-dependent

Thom J, Ivey L, Eikelboom J. Normal plasma mixing studies in the laboratory diagnosis of
lupus anticoagulant. J Thromb Haemost 2003;1:268991

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37C Incubated 1:1 PTT Mix

Patient
+ NP:
incubate
= 1:1 mix:
incubate
1 h, then 1 h, then
plasma
perform perform
PTT PTT

PTT Incubated PTT of Mix

42.5 s PTT 35 s 38.5 s: Correction
Still using 10% prolongation >38.5 s: No correction
as correction cutoff 12
 Mixing Study Result
32-yo Female, 6 Weeks Post-partum
Assay Result RI Comment
PTT 42.5 s 2535 s Confirms previous PTT
PTT/control 1:1 mix Commercial platelet-free
32.1 s Control 30 s
immediate control plasma (NP)
PTT/control 1:1 mix
37.3 s Control 35 s Incubate both 1:1 mix and NP
1 h at 37C
Conclusion: immediate and incubated mix PTTs correct,
suspect factor deficiency, arrange for factor assays and von
Willebrand disease workup

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 Factor Assay Results
32-yo Female, 6 Weeks Post-partum

Assay Result RI Comment

Factor VIII 39%
Factor IX 92% VWD?
50150%
Factor XI 131%
Factor XII 113% XII, HMWK & PK
HMWK deficiency not
ND 65135% associated
PK with bleeding
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 PTT rgt: Ca++, activator, phosphatidyl serine; prolonged by XII, PK,
HMWK, XI, IX, VIII, X, V, II, Fg deficiency; heparin, DTIs, LA

XIa XIIa
VIIa
Extrinsic Pre-K HMWK
TF
IXa
IXa VIIIa
Intrinsic
Figure courtesy of
PT rgt: tissue factor,Ca++, Margaret G. Fritsma,
phosphatidyl serine; prolonged by Xa Va
Rodaks Hematology,
VII, X, V, II, Fg deficiency; direct 5th Edition, 2015
anti-Xa and coumadin Rx
Common Thr XIIIa

Crosslinked
Fibrinogen Fibrin Polymer
Fibrin

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 PT and PTT Test Results
in Inherited Coagulopathies

PT PTT Single Factor Deficiency

Long Normal VII
Long Long X, V, II, and fibrinogen1
Normal Long VIII, IX, XI2
1PT & PTT prolonged when fibrinogen is <100 mg/dL, perform fibrinogen assay
2Contact factor deficiencies XII (13% prevalence), prekallikrein (PK, Fletcher), or

high molecular weight kininogen (HMWK, Fitzgerald) also prolong PTT results, but
no bleeding
 PTT Mix: Why Does This Work?
Hypothetical 20% F VIII level prolongs PTT
PTT rgts calibrated to prolong at 3040% FVIII, IX, XI
Add NP with established 100% factor level
1:1 mix, average of 100% and 20% = 60% (corrects)
Hypothetical anti-FVIII or lupus anticoagulant
With typical avidity, retains ability to prolong the mix

Patient Normal 1:1 Mix:

+ =
60%
20% 100% FVIII
FVIII FVIII
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 Case
52-yo Athletic Female
Pre-op screen for total hip replacement

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 52-yo Athletic Female
Screen Prior to Hip Replacement Surgery

Test Result RI
HGB 14.1 g/dL 1215 g/dL
PT 11.2 s 9.812.6 s
PTT 58 s 2535 s
PLT 170,000/L 150400,000/L
Fibrinogen 410 mg/dL 220498 mg/dL
Patient reports no bleeding or bruising, no thrombosis

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Isolated Prolonged PTT: Differential
Could be nothing: 5% of normals exceed limit
Preanalytical variable: green or lavender-closure
tube, hemolysis, lipemia, clotted specimen
Outpatient: dabigatran
Inpatient: unreported UFH
Congenital single factor deficiency: VIII, IX, or XI,
hemophilia A, B, or C with bleeding, VWD
Congenital FXII, PK, or HMWK without bleeding
FVIII inhibitor (acquired hemophilia) with bleeding
Lupus anticoagulant (LA)
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52-yo Female PTT Mixing Study
Test Result Comment
TT 17 s RI: < 21 s, rules out dabigatran
PTT 58 s RI: 2535 s
PTT NP 28 s Correction if 30.8 s (10%)
1:1 mix 35 s 25% over NP = no correction
What is the next step?

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Acute Care Mixing Study Algorithm
Isolated prolonged PTT

TT long TT

Heparinase TT normal
or polybrene
Pt & NP 1:1 mix No correction
If no correction,
Correction LA profile
dabistop here

Incubated 37C
Correction No correction
Pt & NP 1:1 mix

Factor assay FVIII inhibitor

Consider LA
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 Mixing Study Considerations
Preanalytical variables Not so much
Anti-Xa rivaroxaban, apixaban, edoxaban prolong PT, PTT
Dabigatran and UFH prolong PTT
Mostly
Clotted, hemolyzed, lipemic specimen
Underfilled tube, wrong anticoagulant
PT & NP must be platelet-poor (free), <10,000/uL
Cfg at 2500 g/10 m or double-spin
Heparinase/polybrene neutralize 1 unit/mL UFH
Anti-FVIIIs may generate immediate neutralization
Weak LAs may be missed in 1:1 mix: ask for consult
Select a more LA-sensitive PTT reagent or request 4:1 mix
15% of LAs require incubation 23
 The LA Cofactor Effect
Initial PTT 48 s, RI 2535; 1:1 mix prolongs to 54 s
LA cofactor effect may be prothrombin binds LA
Or maybe LA potentiates clotting via annexin V?
Mix reverses potentiation?

Magrath M. Lupus cofactor phenomenon. Letter J Clin Pathol 1990,42:264.

Rand JH, Wu XX, Andree HA, et al. Antiphospholipid antibodies accelerate plasma coagulation by
inhibiting annexin-V binding to phospholipids: a "lupus procoagulant" phenomenon. Blood.
1998;92:165260.
Clyne LP. Plasma requirement for expression of lupus-like anticoagulant. Folia Haematologica int Ma
Klin Morphol Blutforsch 1986;113:841

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 Normal Plasma Source?
Home brew: ~20 normal plasmas, male female
Ensure plasma is platelet-poor; < 10,000/uL
Ensure NP has ~100% of all factors; PTT MRI
For instance, elevated FVIII causes false negatives
Screen for LA, specific factor inhibitors. HBV, HCV, HIV
Aliquot and freeze
Or purchase commercial plasma
GMP meets all criteria
Frozen meets all criteria
Lyophilized acceptable when validated in house
Processed with stabilizers
Clinical and Laboratory Standards Institute. One-stage prothrombin time (PT) test and activated partial thromboplastin
time test (APTT) approved guidelinesecond edition. CLSI Document H47-A2. CLSI, Wayne PA. 2008.

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 What Limit Defines Correction?
No Consensus; Fritsma Factor Quick Question Results

Limit based on fixed PTT value from reference interval

1:1 mix within RI upper limit (95% or 99% CI, 39%)
1:1 mix within RI upper limit + 5 seconds (8%)
1:1 mix within mean of RI + 2 or 3 SD (0%)
Limits based on NP PTT value
1:1 mix within NP PTT value + 5 seconds (14%)
1:1 mix within NP PTT + 10% (32%)
Limit formula using patient, NP, and 1:1 mix
Must incubate patient sample, NP, and 1:1 mix
Changs % deviation; Rosner index
Combo of RI and Rosner (dedicated RI for mix, 7%)
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 Chang Formula Based on % Correction
Patient PTT 1:1 mix PTT
% Correction = X 100
Patient PTT NP PTT

42.5 32.1 = 10.4

% Correction = = 0.83 = 83%
42.5 30 = 12.5

Factor Deficiency = 75%

Inhibitor = < 75%
% Correction Chang formula verified by local laboratory

Chang SH, Tillema V, Scherr D. A "percent correction" formula for evaluation of mixing studies. Am J
Clin Pathol 2002;117:6273.
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 Rosner Index Based on Ratio

Rosner 1:1 mix PTT NP PTT

= X 100
Index
Patient PTT

Rosner 32.1 30
= X 100 = 4.9
Index 42.5
Inhibitor 11
Correction < 11
Rosner index limit validated by local laboratory
Rosner E, Pauzner R, Lusky A, Modan M, Many A. Detection and quantitative evaluation of lupus
circulating anticoagulant activity. Thromb Haemost 1987; 57: 144-147.
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 59-yo Male
Former Hockey Player

Total knee replacement preop

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 59-yo Male Former Hockey Player
Screen Prior to Knee Replacement Surgery

Test Result RI
HGB 14.8 g/dL 1215 g/dL
PT 11.2 s 9.812.6 s
PTT 38 s 2535 s
PLT 310,000/L 150400,000/L
Fibrinogen 390 mg/dL 220498 mg/dL
Patient reports no bleeding or bruising, no thrombosis

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When to Perform Mixing Study
Any PTT > RI upper limit
Any PTT > RI upper limit + 5 seconds
Any PTT > RI upper limit with consult
Is patient bleeding or clotting?
Possible weak LA: use 4:1 mix
Lupus sensitive PTT reagent
Factor sensitive PTT reagent

Pengo V, Tripodi A, Reber F, et al. Update of the guidelines for lupus

anticoagulant detection. J Thrombos Haemost 2009;7:173740.
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When to Perform Mixing Study
Some Practical Considerations
If you use a value slightly longer than the RI limit and define
correction as return to the RI you miss most inhibitors.
If you perform mixing studies on prolonged PTTs from
inpatients, at least 50% will be due to anticoagulant therapy.
If you call the unit on any prolonged PTT you are likely to get
no information.

Fritsma factor communication, Dr. Emmanuel Favaloro 32

59-yo Male Former Hockey Player

Test Result Comment

TT 17 s RI: < 21 s, rules out dabigatran
PTT 38 s RI: 2535 s
PTT NP 31 s Correction if < 34.1 s (10%)
1:1 mix 35 s Correction? No correction?
What is the next step?

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 59-yo Male Former Hockey Player
Clinical Consult

Consult: if no thrombosis or bleeding, go no further

Thrombosis: perform mix using 4:1 patient to NP
Or choose PTT reagent that is LA-sensitive
If anatomic bleeding symptoms, test FVIII, FIX, FXI
Vitamin K deficiency, renal insufficiency, liver disease,
malignancy, VWD

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2-yo Hemophilic Boy

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2-yo Hemophilic Boy
Test Result RI
HGB 11.8 g/dL 9.615.6 g/dL
PT 11.2 s 9.812.6 s
PTT 65 s 2535 s
PLT 310,000/L 150400,000/L
Fibrinogen 390 mg/dL 220498 mg/dL
Inflamed, swollen knee and ankle

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 Mixing Study Result
2-yo Hemophilic Boy

Assay Result RI Comment

PTT 65 s 2535 s Confirms previous PTT
PTT/control 1:1 mix
33.5 s Control 30 s Correction
immediate
PTT/control 1:1 mix Control is incubated
47.9 s Control 35 s
1 h at 37C alone and with mix
Conclusion: Anti-FVIII inhibitor

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 Factor VIII Assay
Dilute plasma 1:10, add FVIII-depleted rgt plasma 1:1
Add PTT reagent, incubate 3 minutes
Add CaCl2, record interval to clot formation
Compare result in seconds to dilution curve
Factor VIII Activity Reference Curve

1000

100
Seconds

10

1
1 10 100
Pe rce nt Activity
38 38
Factor VIII Assay Dilutions Parallelism
Indicates No Inhibitor

Plasma Dilution Seconds Raw Factor VIII Computed Factor VIII

Activity Activity ( dilution)
1:10 undiluted 90 s 20% 20%
1:20 104 s 10% 20% (parallel)*
1:40 107 s 5% 20% (parallel)
1:80 110 s 2.5% 20% (parallel)
* <10% difference from undiluted indicates parallelism, no inhibitor

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 FVIII Assay Dilutions
non-Parallelism Indicates Inhibitor
Plasma Dilution Seconds Raw Factor VIII Computed Factor VIII
Activity Activity ( dilution)*
1:10 undiluted 80 s 10% 10%
1:20 93 s 8% 16%
1:40 107 s 5% 20%
1:80 108 s 4% 32%
* >10% difference from undiluted, rising = non-parallel, implies inhibitor

Kasper CK. Laboratory diagnosis of factor VIII inhibitors. In Kessler C, Garvey MB, Green D, Kasper C, Lusher
J. Acquired Hemophilia 2nd Edition. Excerpta Medica 1995
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55-YO Male with
Atrial Fibrillation

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55-yo Male with Atrial Fibrillation

Test Result RI
HGB 13.8 g/dL 1215 g/dL
PT 17.2 s 9.812.6 s
PTT 159 s 2535 s
PLT 310,000/L 150400,000/L
Fibrinogen 20 mg/dL 220498 mg/dL

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55-yo Male with Atrial Fibrillation

Assay Result RI
PTT 159 s 2535 s
TT > 150 s < 21 s
PTT/control 1:1 mix
78 s Control 30 s
immediate
PT/control 1:1 mix
15.2 s Control 12 s
immediate
What do you recommend?

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 If the PT is Prolonged
Congenital deficiencies of II, V, VII, or X
PT and PTT long: II, V, X
PT only: VII, skip mixing and go to factor assay
Prevalence: 500,0001:2,000,000
Liver disease: PT prolongs before PTT due to
des-carboxy II, VII, and X, reduced factor V
Vit K deficiency: des-carboxy II, VII, and X
Anti-Xa direct oral anticoagulants
Rivaroxaban, apixaban, edoxaban
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Isolated Prolonged PTT: Summary
Random benign prolongation, 95% CI
Lupus anticoagulant: 13%
Drug reaction producing transient LA
Unrecorded heparin, dabigatran, oral anti-Xa
Known hemophilic who fails FVIII concentrate Rx
Hemorrhage or ecchymoses signal acquired coagulopathy;
vitamin K deficiency, liver disease
Specific inhibitor, anti-FVIII: post partum, malignancy,
autoimmune disorders, > 60 YO
Sahud MA. Factor VIII inhibitors. Laboratory diagnosis of inhibitors Semin Thromb Hemost 2000;26:195
203.
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Develop Mixing Study Reliability

Test PTT reagent sensitivities

3040% FVIII, FIX, FXI
Select Intermediate sensitivity to LA
NP consistency: ~100% activity for all factors
Consultation for equivocal patient results
Employ consistent correction limit

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 Perform Mixing Studies Locally

Unexpected isolated prolonged PTT or PT

requires immediate action
Delay results in specimen deterioration
Perform locally, results may immediately direct
therapy
Forward results to ref lab to direct follow-up

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 Summary: Mixing Studies
at the Acute Care Facility

The participant
1. Prepares a stepwise PTT and PT mixing study protocol
2. Indicates the clinical purpose for PTT mixing studies
3. Explains why the mixing study is an acute care assay
4. Correlates mixing study results with coagulation test results
Precision BioLogic Inc
140 Eileen Stubbs Avenue
Dartmouth, NS B3B 0A9
precisionbiologic.com
US& CA: +1.800.267.2796 48
Thanks for listening!

Questions?

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