1. Innovations in Coagulation Testing:
State of the Art of Coagulation
Testing
ELLINOR I. PEERSCHKE, PH.D., F.A.H.A.
VICE CHAIR, LABORATORY MEDICINE
HEAD, HEMATOLOGY & COAGULATION LABORATORY SERVICES
MSKCC

2. Overview
Discuss performance characteristics of screening
tests, and utilization of the APTT
Discuss the state-of-the art of current coagulation
test performance
 F VIII, F IX, XI
 Lupus Anticoagulant Detection
 Case Studies

3. Hemostasis Studies
Types of Assays
 clot based
 chromogenic
 immunologic assays

4. Screening Tests
PT
APTT
Fibrinogen
Thrombin Time
 Heparin Contamination
 Hypo/Dysfibrinogenemia (follow with Reptilase Time)
 Need fibrinogen result for interpretation!

5. APTT
1954 (K.M. Brinkaus et al.) developed a
recalcification time assay
+ Platelet substitute = Partial thromboplastin time
 Rabbit brain
 Newer assays use soy based synthetic phospholipids
+ negatively charged activators = APTT
 Better reproducibility
 Kaolin, silica, ellagic acid, celite
 Colloidal silica

6. Selection of APTT Reagent
Screen for Factor Deficiency
Monitor Heparin
Detect Lupus Anticoagulant
Specialized Testing: Factor Assays
A single reagent may not be able to fulfill all
requirements!

7. APTT Methods
Clot based method
 Original
 Technologist + water bath+ stopwatch
 Current Automated
 Optical
 Mechanical

8. Sensitivity of Screening Tests
PT/APTT : prolonged by single factor deficiency <30%
(variable)
Upper limit reference range : 30 sec
PT: highly sensitive to multiple Vitamin K dependent
factor deficiencies; monitor warfarin anticoagulation
with INR
APTT: sensitive to heparin
 Heparin therapeutic range (0.3 – 0.7 IU/ml)
 sensitive to LMWH

9. Screening APTT Reagent Selection
Do we want to detect a lupus anticoagulant during
routine APTT screening?
Consequences of using a screening APTT reagent
with moderate sensitivity to LAC
 Delay in patient care (surgery, invasive procedures) until factor
deficiency is excluded
 Expense
 Hospital
 Laboratory

10. Case Study
49 y.o. female with cardiomyopathy and stage D
systolic heart failure – requires a biventricular
pacemaker
Screening APTT 132.3 sec (23.6 -35.7 sec)
Stago/Roche STA PTT A reagent
Thrombin Time: 17.6 sec (<21 sec)
no heparin contamination
No Bleeding History

11. Mixing Study
1:1 Mix Patient Plasma: PNP
 APTT 58 sec
 APTT PNP 28 sec
 Interpretation: Partial Correction
 Circulating anticoagulant; Factor deficiency

12. Additional Studies
Reflex APTT with LAC insensitive reagent
Dade Behring Actin FS: 31.1 sec
(reference range 23.2 -32.0 sec)
Interpretation:
No significant factor deficiency
High likelihood of lupus anticoagulant
LAC confirmed by StaClot LA

13. APTT Reagents
Reagent Lupus Anticoagulant
Sensitivity
Siemens Actin FSL Sensitive
Hemosil APTT-SP
Stago STA-PTT A Moderate
Hemosil Synthasis
Siemens Actin FS Insensitive
Stago CK Prest

14. Lupus Anticoagulant Sensitivity of APTT reagents used by laboratories
subscribed to CAP proficiency surveys
4500
Number of Laboratories
4000 2
3500
3000
2500 1
2000
1500
1000 3
500
0
Sensitive Moderate Insensitive
Reagent Sensitivity to Lupus Anticoagulant
1= Siemens Actin FSL 2= Diag Stago STA-PTT A
Hemosil APTT-SP Hemosil Synthasil
3=Siemens Actin FS

15. Survey of Clinical Coagulation Laboratories
Evaluate Coagulation
Laboratory Practices  Survey design:
 APTT reagents &  George Fritsma,
 The Fritsma Factor
utilization
 Ankush Randhawa
 Sponsored in part by  Precision Biologic
Precision Biologic &  Dr. Marissa Marques
Fritsma Factor  University of Alabama,
Dialog about best Birmingham
 Dr. Dorothy Adcock
practices  Esoterix, Denver, CO
 Meeting clinical  Dr. Elizabeth Van Cott
expectations  Mass General Hospital,
Boston, MA
 Sponsored by Stago

16. Respondents
Supervisor
Manager
Pathologist
Lab Director Technologist
Lead Technologist
Technical Specialist
N=93

17. Daily APTT Volume for Responding Laboratories
APTT volume Laboratories (#)
> 300 32
150-300 15
10-80 45

18. APTT Utilization
Indication % of local APTT
volume
Monitor Unfractionated 49%
heparin therapy
Screen for Coagulation 41%
Factor Deficiency
Screen for Lupus 14%
Anticoagulant

19. What is the lupus anticoagulant sensitivity level of your high-
volume routine screening APTT reagent?
HIGH
Moderate
LOW
Don’t know

20. What is the lupus anticoagulant sensitivity level of your
high-volume routine screening APTT reagent?
N=93 responses

21. Does a normal APTT rule out a lupus anticoagulant?
YES
NO
Don’t Know

22. A normal APTT rules out a lupus anticoagulant.

23. Sensitivity of Screening APTT to Lupus
Anticoagulant
NASCOLA 2008 surveys
 20% False Negative on sample with low titer LAC (n=58)
MSH – (Stago Roche PTT Automate)
~25% False Negative
(normal APTT with positive LA work-up)
 DRVVT
 StaClot LA

24. Detection of Lupus Anticoagulant
ISTH guidelines 2009
 Perform at least 2 screening tests which demonstrate
prolongation of a phospholipid-dependent clotting time using
different testing principles (APTT, DRVVT)
 Perform mixing studies to confirm the presence of a circulating
anticoagulant and rule out factor deficiency
 Perform confirmatory tests that demonstrate phospholipid
dependent inhibitory activity

25. Why do laboratories chose reagents with LA sensitivity?
Instrument – reagent compatibility
 QA programs
 Troubleshooting test performance issues
Need for larger commercial reagent portfolio

26. Approach to abnormal
PT or APTT
Factor Deficiency Mixing Studies
Circulating anticoagulant 1:1 Immediate Mix
 Lupus anticoagulant  Patient Results
 acquired inhibitors  PNP
 F VIII, F V ( F II)  Patient : PNP
 Paraproteins Interpretation: What is
 Anticoagulants
correction?
Factor deficiency(ies) vs.
lupus anticoagulant
 Repeat APTT with LAC
insensitive reagent

27. Abnormal APTT work-up
Partial/No Correction of 1:1 Mixing Study
LA studies & Factors VIII, IX, XI
 if LA neg, normal factors:
 continue with F XII, PK, HMWK
If LA positive with normal Factors
 check PT
 if abnormal, perform F II assay

28. Factor Assays: Interlab Variability
(NASCOLA PT/EQA Results)
2003 Proficiency Testing Program
Survey ID Factor Method Mean (%) Range (%) CV (%)
01-03 VIII Clot (n= 97) 23 12-36 20%
Chrom 22 17-27 20%
(n= 4)
02-03 Clot 80 50-116 14%
(n= 101)
Chrom 79 68-94 14%
(n= 4)
IX Clot 57 38-83 14%
(n= 111)
23 12-38 22%
02-03 XI Clot 55 32-70 14%
(n= 107)
84 58-117 14%

29. Evaluation of Severe Deficiencies
F VIII , 1.0 %
 CV high
 Recommend extended curves
 Use reference plasma that is calibrated against a WHO
standard

30. High Factor Level: Patient Plasma
Factor VIII
# Reporting 51
Labs
High Factor VIII
Expected 178
Result (%)
Mean 188% oCV high
Range 134-
280% oRecommend extended curve
CV 16%
Classification oUse reference plasma that is calibrated to
WHO standard
Normal 57%
Borderline 2%
Normal
Borderline
Abnormal
Abnormal 34%

31. Intrinsic Pathway Factor Assay Variables
Assay Type
 clot based
 One stage assay
 chromogenic assays
Activator
Deficient Plasma
Calibrator Plasma
Equipment

32. Intrinsic Pathway Factor Assays: Assay
Conditions (NASCOLA)
 Activator (14- 15different activators used)
 24% Stago/Roche PTT Automate
 20% Siemes/Dade Behring Actin FSL
 14% Siemes/Dade Behring Actin FS
 Deficient Plasma ( 8-10 different deficient plasmas used)
 25% Precision Biologics
 20% George King
 16% Siemens Dade Behring
 16% HRF Inc.
 Calibrator (9-10 different calibrators used)
 27% Precision Biologic Normal Reference Plasma
 27% Stago Roche Unicalibrator
 20% Siemens Dade Behring Standard Human Plasma
 12% I.L. Calibration Plasma
 Equipment (7-10 different analyzers used)
 30% Stago Roche STA, STA Compact, STA-R
 22% Siemens Dade Behring BCS
 12% Stago/Roche STA-R Evolution
 12% MDA BioMerieux
 10% Abbott IMx

33. Impact of Assay Variables on Factor Results
Variable Data Sets % Data Sets with Findings
(from D. D. Castellone, (n)*
Analysis of APTT Reagent/Activator
ECAT/NASCOLA
Ellagic Acid 96 32% Inter laboratory CV >20%
proficiency testing data
Micronized Silica 128 31% Mean factor level 20% below target
for factors VIII, IX, XI,
XII from 2003-2007) Cephalin Silica 128 16% Mean factor level 10-15% above target
Clot Detection Method
* Participant results Electromechanical 96 33% Inter laboratory CV >20%
submitted for each factor
Optical 96 40% Inter laboratory CV >20%
VIII, IX, XI, and XII assay
challenge represented one Reference Plasma Standard
data set Fresh Frozen 96 29% Inter laboratory CV >20%
Lyophilized 96 40% Inter laboratory CV >20%
Deficient Plasma Source
Congenitally Deficient 96 35% Inter laboratory CV>20%
Immunoabsorbed 96 53% Inter laboratory CV >20%

34. Performance of Major LAC Screening Tests:
NASCOLA
ASSAY 2008-1 2008-2 2008-3 2008-4 2009-2
Medium Titer LAC
High Titer LAC Plasma Medium Titer LAC Low Titer LAC Plasma
Plasma Normal Plasma Pool
Pool Plasma Pool
(Diluted)
False Negative False Negative False Negative False Negative False Positive
All 0% 0% 5.9% 9.6% 6.6%
APTT (combined) 0% 0% 4.5% 2.4% 5.4%
APTT (LAC sensitive) 0% 0% 3.0% 3.2% 7.4%
APTT (LAC moderate 0% 0% 7.1% 0% 0%
sensitivity)

35. Performance of Confirmatory Assays: NASCOLA
ASSAY 2008-1 2008-2 2008-3 2008-4 2009-2
Intermediate Titer
High Titer LAC Intermediate Titer Low Titer LAC
LAC Plasma Normal Plasma Pool
Plasma Pool LAC Plasma Plasma Pool
(Diluted)
False Negative False Negative False Negative False Negative False Positive
Integrated- APTT based
0% 82% 5% 89% 31%
dRVVT
3% 29% 26% 62% 5%

36. Overall State of the Art of Hemostasis Testing
in North America
o Test peformance characteristics have not changed
significantly since 2003
o Imprecision of assay results remains high
o Will greater STANDARDIZATION improve
performance? reagents
test systems
testing algorithms
interpretive reporting

37. 75 y F – ICU - Pneumonia
PT 18.5 s (12.2 – 13.5s)
APTT 220s (24.8 – 35.5s)
Fibrinogen 334 mg/dl (180 – 400 mg/dl)
D-dimer: 5.4 µg/ml (< 1.1 µg/ml)
Plt: 136K/µl (150 – 450 K/µl)

38. 75y F- ICU
Consider:  PT 18.5 s
DIC (12.2 – 13.5s)
 APTT 220s
lupus anticoagulant with
F II deficiency, (24.8 – 35.5s)
 Fibrinogen 334 mg/dl
 Liver dysfunction,
(180 – 400 mg/dl)
Vit K deficiency,  D-dimer: 5.4 µg/ml
heparin contamination (< 1.1 µg/ml)
 Plt: 136K/µl (150 – 450
K/µl)

39. 75y F- ICU
Mixing Study
 Patient APTT 156s
 Pooled Normal Plasma 30.7 s
 1:1 immediate mix 113.2
 Patient PT 18.8s
 Pooled Normal Plasma 11.4 s
 1:1 immediate mix 14.2 s
No Correction: Circulating Anticoagulant Present
o APTT Actin FS: 120s (normal 23.2 -32.0 sec)

40. 75y F- ICU
 Thrombin Time
 >300 sec (normal <21 sec)
 Reptilase Time
 22 sec (normal < 21 sec)
 Factor Assays
 F II 83%
 F V 121%
 F VII 114%
 Heparin contamination, no evidence of Vit K deficiency,
or acute liver dysfunction
 ? Prolonged PT

41. 81 y F -ICU - Bleeding after colonoscopy
PT 12.6 s (12.2 – 13.5s)
Immediate Mix: APTT 48 s (24.8 – 35.5s)
 PNP 28.7 s
 1:1 mix 32.0 sec
Incubated Mix: APTT 51 s
 PNP 30.2 s
 1:1 mix 50 s
APTT Actin FS 39 sec (normal 23.2 -32.0 sec)

42. 81 y F - ICU - Bleeding after colonoscopy
 F VIII 12%
Suspect acquired F VIII inhibitor
 Bethesda Titer 1.8 units
NB: F VIII inhibitors are often not detected with
immediate mixing study