Laboratory testing for bleeding disorders: Strategic uses of high and ...

Laboratory testing for bleeding disorders: 

Strategic uses of high and low yield tests 

Catherine P. M. Hayward, MD PhD, FRCP(C) 

Head, Coagulation, Hamilton Regional Laboratory Medicine Program 

Professor, Pathology and Molecular Medicine 

McMaster University, Hamilton, Ontario, Canada

Disclosures and Acknowledgments 

for Catherine P. M. Hayward 

• Nothing to disclose 

• Acknowledgment of materials from: 

1) Hayward, Moffat. Int J Lab Hematol. 2013 epub Mar 11 

2) Hayward, Moffat, Liu. STH 2012;38(7):742-52. 

3) Hayward, Moffat, Plumhoff, Timleck, Hoffman, 

Spitzer, Van Cott, Meijer. STH 2012;38(6):622-31 

4) Other sources cited in slides

Illustrative Case 

• 51 year old male 

• Recent severe, unexplained retroperitoneal 

bleed (ICU stay) + many serious bleeding 

episodes (all delayed in onset) when challenged 

previously. 

• Prior referrals to 5 hematologists, who excluded: 

▫ Hemophilia, other factor deficiencies (including 

factor XIII and fibrinogen disorders) and von 

Willebrand disease 

• What tests should be ordered now?

Perspectives on Bleeding Disorder Diagnosis 

• Laboratory tests are essential for diagnosis 

• Tests and panels offered by labs are not 

standardized 

▫ Most offer coagulation screening tests in bleeding 

disorder panels, without complete testing for more 

common defects in primary hemostasis 

▫ Few offer tests for rare bleeding disorders 

• Strategies to guide investigations (including 

when to order tests for rare bleeding disorders) 

are rarely provided by laboratories

Coagulation Screening Tests in 

Bleeding Disorder Investigations 

• Detect some important disorders 

• Problems: 

▫ False positives and false negatives do occur 

▫ More specific tests (e.g., factor assays) are required to: 

• Detect mild factor deficiencies 

including hemophilias from unstable factor VIII 

• Further assess abnormalities

Tests for Defects in Primary Hemostasis 

• Much greater sensitivity for common bleeding 

disorders than coagulation screening tests 

• Pitfalls: 

▫ “Undefined bleeding disorders” 

• Similar bleeding history to von Willebrand disease 

and platelet function disorders but normal test 

findings 

▫ Criteria used to define “abnormal” 

• e.g., Cutoffs for von Willebrand disease screens

Tests for Other Bleeding Disorders 

• Essential to diagnose some conditions, 

particularly those with delayed-onset bleeding, 

such as: 

▫ Factor XIII deficiency 

▫ α2 antiplasmin deficiency 

▫ Plasminogen activator inhibitor-1 deficiency 

▫ Quebec platelet disorder

Other Considerations: Pretest Probabilities 

• Patients in preoperative clinics 

▫ Low pretest probability for a bleeding disorder 

• Patients seen for bleeding disorder evaluation 

▫ Much more likely to have a bleeding disorder 

• Family history of bleeding disorder 

▫ Relatives are at higher risk 

• Population/social factors 

▫ Affect bleeding disorder prevalence 

▫ Consanguinity: culturally accepted in some populations 

‣Higher prevalence of rare bleeding disorders

Other Considerations: Abnormalities Can Reflect 

• Age related changes in hemostasis 

• False positives or true positives that are not 

clinically important (e.g., contact pathway factor 

deficiency) 

• True positives from drugs (e.g., anticoagulants, 

drugs that inhibit platelet function) 

• True positives from clinically important, 

congenital or acquired bleeding problems 

• ADDITIONAL CHALLENGE: interpretation

CAUSE AND PATTERN OF ABNORMALITIES PT/INR APTT TT FIBRINOGEN 

Fibrinogen deficiency (hypofibrinogenemia) or dysfunction 

(dysfibrinogenemia) 

N – ↑ N – ↑ ↑ ↓ 

Afibrinogenemia NC NC NC ND 

FVII deficiency ↑ N N N 

FVIII, FIX , and/or FXI deficiency N ↑ N N 

Acquired or congenital hemophilia, with an inhibitor N ↑ † N N 

FII, FV, and/or FX deficiency ↑ ↑ N N 

Factor deficiencies not associated with bleeding (FXII, high molecular 

weight kininogen or prekallikrein deficiency) 

N ↑ N N 

Lupus anticoagulant N – ↑ N – ↑ ‡ N N 

Lupus anticoagulant with FII deficiency ↑ ↑ N – ↑ N 

Unfractionated heparin - therapy or sample contamination N – ↑ ↑ ↑↑ * N 

Low molecular weight heparin therapy N N – ↑ N – ↑ N 

Direct thrombin inhibitors N – ↑ N – ↑ ↑↑ N 

Direct inhibitors of FXa N – ↑ N – ↑ N N 

Liver disease † (if early, often affects FVII, FXI and/or FXII; if late or end 

stage, fibrinogen is usually low; spares FVIII but can affect all other 

factors) 

N – ↑ N – ↑ N – ↑ ↓ - N – ↑ 

Vitamin K deficiency (or treatment with a vitamin K antagonist) which 

reduce levels of FVII and also FII, FIX and FX † 

↑ N – ↑ N N 

Fibrinolytic therapy ↑ ↑ ↑ ↓ 

Consumptive coagulopathy † N – ↑ ↑ N – ↑ N – ↓ 

Dilutional coagulopathy † N – ↑ N – ↑ N – ↑ ↓ - N 

VWD N N – ↑ N N 

Preanalytical error – collected in potassium EDTA § ↑ ↑ N – ↑ N 

Preanalytical error – serum instead of plasma NC NC NC ND 

N, normal; NC, no clot; ND, not detected

Evidence on Test Sensitivity and Specificity for 

Bleeding Disorder Investigation? 

• Recent research 

▫ Hamilton Regional Laboratory Medicine Program 

(HRLMP) 

• Center that tests local and referred samples for common and 

rare bleeding disorders 

• Approach 

▫ 1) Estimated sensitivities and specificities of lab tests 

for bleeding disorder diagnosis from a prospective 

cohort study (Hayward, Moffat, Liu. STH 2012;38(7):742-52) 

▫ 2) Assessed findings for rare bleeding disorder tests by 

a retrospective review of records (Jan 2003- Dec 2012; 

Hayward, Moffat. IJLH current issue)

Sensitivity and Specificity for Bleeding Disorders 

standard investigations done so this comparative analysis was possible 

Hayward, Moffat, Liu. Semin Thromb Hemost. 2012;38(7):742-52 

Test Sensitivity Specificity 

APTT 3.1% 98% 

PT/INR 0.5% 100% 

Fibrinogen 1.0% 100% 

Thrombin Time 1.0% 98% 

VWD Screen 6.0% 98% 

LTA* 25.7% 98% 

*LTA performed and interpreted in accordance with North American Guidelines 

Hayward et al, Am J Clin Pathol 2010;134:955-963

Test 

APTT 

PT/INR 

Factor 

assays & 

factor 

inhibitors 

Abnormalities associated 

with bleeding 

Congenital or acquired 

deficiencies of intrinsic 

(FVIII, FIX and FXI) and 

common pathway factors 

(FII, FV and FX) that cause 

bleeding 

Congenital or acquired 

deficiencies of factor VII 

and common pathway 

factors (FII, FV, and FX) 

Congenital and acquired 

deficiencies of coagulation 

factors II, V, VII, VIII, IX, X 

and XI. 

Practice Points 

Abnormalities 

unrelated to bleeding 

(excl. preanalytical) 

Contact factor 

deficiencies (FXII, 

prekallikrein, high 

molecular weight 

kininogen) and most 

lupus anticoagulants 

Considerations 

• Most abnormalities are not 

clinically significant 

• Does not detect some mild factor 

deficiencies and most fibrinogen 

disorders 

• Requires follow up factor assays 

• Does not detect some mild factor 

deficiencies and most fibrinogen 

disorders 

• Requires follow up factor assays 

• Anticoagulants and lupus 

anticoagulants can interfere 

• Two stage (or chromogenic) 

assays may be required to detect 

hemophilia due to unstable FVIII 

• Unknown sensitivity/specificity


Test 

Thrombin 

time (TT) 

Abnormalities 

associated with bleeding 

Congenital and acquired 

defects in fibrinogen 

(quantitative and 

qualitative) and fibrin 

polymerization, including 

elevated fibrin(ogen) 

degradation products 

Abnormalities 

unrelated to 


Valproic acid therapy 

and some 

paraproteins can 

prolong the test, as 

does heparin and 

direct thrombin 

inhibitors 


• Correlate with fibrinogen level 

• Measure fibrinogen antigen and 

reptilase times if a congenital 

defect is suspected 

• Acquired abnormalities are more 

common 

Clauss 

fibrinogen 

Quantitative 

(afibrinogenemia, 

hypofibrinogenemia) and 

qualitative 

(dysfibrinogenemia) 

defects in fibrinogen 

_ 

• Correlate with thrombin time 

• To distinguish quantitative from 

qualitative defects, consider testing 

TT of control diluted to the same 

fibrinogen level 

• If abnormal, and congenital 

deficiency is suspected, test 

fibrinogen antigen, TT and reptilase 

times

Illustrative Findings: Fibrinogen Disorders 

From Verhovsek, Moffat, Hayward. Am J Hematol 2008;83(12):928-31 

Test RI Hypofibrinogenemia Dysfibrinogenemia Afibrinogenemia 

Clinical problem 

Recurrent pregnancy 

loss from abruptions 

without any other 

bleeding symtoms 

Menorrhagia, PPH 

requiring transfusion. 

Intracranial 

hemorrhage, 

traumatic & surgical 


PT 11-14 s 14.2 13.0 No Clot 

INR 0.8-1.2 1.2 1.1 No Clot 

APTT 22-35 s 32 33 No Clot 

TT 20-30 s 46 122 No Clot 

Reptilase time 

15-27 s 

Clauss Fbgn 

1.6-4.2 g/L 

Fbgn antigen 

1.6-4.2 g/L 

33 40 >60 

0.6 1.2

Test 

VWD Screen 

(FVIII, 

VWF:Ag, 

VWF:RCo; 

multimers if 

abn. found) 

LTA 

Abnormalities 

associated 

with bleeding 

Congenital and 

acquired VWD 


acquired 

platelet 

function 

defects 


Abnormalities 

unrelated to 


Low VWF:RCo in 

some African 

Americans with 

polymorphisms that 

don’t impair VWFplatelet 

receptor 

interactions 

Single agonist 

abnormalities are 

often false positives 

(exceptions: 

collagen, ristocetin) 


• Cutoffs used in practice vary, 

which influences the 

sensitivity and specificity 

• Levels influenced by age, 

blood group, menstrual 

cycle, oral contraceptives, 

exercise, illness and stress 

• Normal in some platelet 

disorders (including some 

dense granule deficiencies 

and release defects) 

• May be falsely normalized 

by lumiaggregometry 

reagent


Test 

Lumiaggregometry 

assay of 

platelet dense 

granule ATP 

release 

Electron 

microscopy for 

platelet dense 

granule 

deficiency 

Abnormalities 

associated with 



acquired defects 

in dense granule 

release, a 

common type of 

platelet function 

disorder 


acquired platelet 

dense granule 

deficiency 

Abnormalities 

unrelated to 


False positives 

can reflect 

preanalytical 

errors 

False positives 

are rare and 

can reflect 

preanalytical 

errors 

(platelet 

activation) 


• Usually a secondary 

investigation 

• Release with weak agonists is 

variable 

• Reagent can falsely normalize 

some aggregation findings 

• Sensitivity and specificity for 

bleeding disorders not reported 

• Usually a secondary 

investigation 

• Detects only one kind of 

platelet disorder 

• We test if dense granule release 

is reduced with strong agonists 

• Specificity >99%, sensitivity for 

bleeding disorders not reported

Realities of Practice 

• Laboratories have greater difficult with aggregometry 

interpretation than with assessing findings of electron 

microscopy tests for platelet dense granule deficiency 

• Performance is particularly problematic for interpreting 

common aggregation abnormalities 

Hayward, Moffat, Plumhoff, Timleck, Hoffman, Spitzer, Van Cott, 

Meijer. STH 2012;38(6):622-31

Test 

Sensitivities and Specificities 

Comparisons of Bleeding Disorder Panels 

Hayward, Moffat, Liu. STH 2012;38(7):742-52 

Sensitivity Specificity 

APTT, PT/INR, Fibrinogen, Thrombin Time 3.0% 98% 

APTT, PT/INR, Fibrinogen, Thrombin Time 

& VWD Screen 

APTT, PT/INR, Fibrinogen, Thrombin Time, 

VWD Screen & LTA 

7.8% 95% 

29% 93% 

Practice points: 

1. Tests for primary hemostatic defects are needed for the panel to have 

reasonable detection of common bleeding problems (factor deficiencies are 

important but less common) 

2. Some disorders are diagnosed without these tests (e.g. aspirin-induced 

bleeding, ITP, etc.) or by other tests (e.g., disorders of fibrinolysis) 

3. Doing VWD screens and LTA at the same time reduced false negatives

55 year old lady, very positive bleeding history & two 

children with VWD confirmed by genetic testing 

Patient 

RI 

PT/INR 1.1 INR 0.8 – 1.2 INR 

APTT 33 sec 22 – 35 s 

Factor VIII 0.80 U/mL 0.50 – 1.50 U/mL 

VWF:RCo 0.59 U/mL 0.50 – 1.50 U/mL 

VWF:Ag 0.61 U/mL 0.50 – 1.50 U/mL 

VWF multimers 

Normal 

distribution

LTA findings 

%MA %MA 

Patient RI 

ADP 2.5 µM 31 >24 

ADP 5.0 µM 87 >43 

Collagen 1.25 µg/mL 87 >51 

Collagen 5.0 µg/mL 89 >85 

Epinephrine 6 µM 91 >9 

Arachidonic Acid 1.6 mM 88 >77 

Thromboxane analogue U46619 1 µM 89 >70 

Ristocetin 0.5 mg/mL 82/84/80 75 

Type 2B VWD confirmed by genetic testing

Test 

Tests for Rare Bleeding Disorders 

% abnormal among 

evaluated patients 


Quantitative 

factor XIII 

activity assays 

Factor XIII 

antigen assays 

comments 

• Low in 12.7% 

patients evaluated 

by HRLMP 

• Lowest in severe 

FXIII deficiency 

(congenital and 

acquired cases) 

• Common: mild 

deficiencies in sick, 

hospitalized 

patients 

• A subunit antigen 

results show strong 

correlation with 

activity 

• No sample blank in kit; most samples with 

0.07-0.13 U/ml (lower RI limit: 0.60 U/ml) 

had undetectable FXIII by urea clot solubility 

• Useful to follow therapy (e.g., with 

pregnancy, surgical interventions). 

• Detects some mild deficiencies (e.g., carriers 

among relatives of severe deficient patients) 

missed by clot solubility assays 

• CAUTION: mild deficiencies in sick 

hospitalized patients do not appear to be 

predictive of bleeding 

• Most assays measure A subunit (B subunit 

assays are presently not available) 

• Subunit A (enzymatic) >>>> subunit B 

deficiency (no known B subunit deficient 

patients in Canada) 

• Perform if there is low activity

Factor XIII Antigen Levels 

see Poster 349 (board 79) by Moffat et al 

FXIII subunit B (U/ml) 

2.0 

1.8 

1.6 

1.4 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 

FXIII subunit A (U/ml) 


1. Severe deficiency commonly affects subunit A 

2. Subunit B can be reduced or normal when subunit A is deficient

FXIII activity (U/ml) 

1.8 

1.6 

1.4 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

Factor XIII Activity Compared to Antigen Levels for Subunits 

see Poster 349 (board 79) by Moffat et al 

FXIII activity (U/mL) 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0.0 

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 

FXIII subunit A (U/ml) 

0 

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 

FXIII subunit B (U/mL) 

Practice point: 

1. While Factor XIII circulates as a complex of A and B subunits, subunit A (which is 

the enzymatic component) shows stronger correlation with activity than subunit B


Test 

Lower 

reference 

interval limit 

α 2 antiplasmin 

(α 2 AP) 

0.80 U/mL 

% abnormal among evaluated 

patients 

comments 

• Low in 6.4% of tested patients 

• Lowest: congenital deficiency 

(0.18-0.23 U/mL) 

• Several suspected heterozygous 

deficiencies (milder but 

persisting deficiency + delayed 

bleeding symptoms without 

other causes) 

• Many abnormalities reflect 

acquired deficiency from 

consumption: 0.30-0.79 U/mL 


• Homozygous 

deficiency: rare 

• Heterozygotes: milder, 

not all are symptomatic 

• Consumed in DIC and 

other conditions with 

elevated D-dimer 

• No evidence that 

measuring α 2 AP is 

helpful in consumptive 

states


Disorder or Test 

ELT 

Euglobulin clot lysis 

time 

(clot made by adding 

thrombin to a 

redissolved 

precipitate of plasma 

acid-insoluble 

globulin) 

% abnormal among 

evaluated patients 

comments 

• Short ELT are not that 

common ~1.8% of patients 

tested; many had a 

documented acquired 

coagulopathy 


• NOT a global test for fibrinolytic 

disorders (e.g., normal in QPD, 

α 2 AP deficiency) 

• Elevated PAI-1 prolongs ELT 

• Should do if: 1) fibrinogen is 

normal and 2) severe, congenital 

PAI-1 deficiency is suspected 

• ELT: notably short in severe PAI-1 

deficiency (very rare) 

Plasmin activator 

inhibitor-1 (PAI-1) 

assays 

• No deficiencies 

• Activity: no lower RI limit (assay 

designed for measuring high 

levels; declining interest in 

measuring increased levels) 

• Antigen: can help diagnose 

deficiencies

Back to Initial Case with an Undiagnosed Bleeding Problem 

Result 

Reference Interval 


time 

(assess for PAI-1 

deficiency) 

>6.0 (prolonged) 1.5 – 5.5 hrs 

α 2 antiplasmin (α 2 AP) 0.18-0.23 

(multiple determinations)* 

0.80 – 1.20 U/mL 

*His children had asymptomatic, milder α 2 AP deficiency (carriers) 


1. Differential diagnosis of life long, bleeding problems includes 

congenital disorders of fibrinolysis, which should be suspected when 

the history indicates delayed-onset bleeding after trauma/surgery and 

factor deficiencies have been excluded 

2. When making decisions on testing, one needs to consider what was 

not excluded by prior investigations

Concluding Practice Points 

laboratory diagnosis of bleeding disorders 

• Current tests for bleeding disorders have high 

specificity 

• The different sensitivities of tests for bleeding 

disorders reflect differences in disorder 

prevalence amongst patients referred for bleeding 

assessment 

• Some tests are of questionable value for an 

evaluation of an acquired coagulopathy 

• Consider testing strategies, and when to order 

tests for rare bleeding disorders (e.g., next slide)

Initial Panel for Bleeding Disorder Investigations 

PT/INR, APTT, TT, fibrinogen, VWD screen, LTA, complete blood count 

Coagulation tests abnormal 

If fibrinogen and TT abnormal, 

consider hypo- and dysfibrinogenemia, 

valproic acid effect, direct thrombin 

inhibitor treatment, DIC. 

Isolated, abnormal APTT: exclude 

clinically significant factor (factors VIII, 

IX, and XI deficiencies). Consider 

other causes: factor XII deficiency 

(other contact factor deficiencies) , 

lupus anticoagulant and heparin 

If low factor VIII, review VWD screen, 

consider inhibitor testing 

PT/INR and APTT abnormal: test to 

determine if there is single or multiple 

factor deficiency (e.g. from vitamin K 

deficiency, vitamin K antagonists, liver 

disease, combined factor V and VIII 

deficiency) 

Isolated, abnormal PT/INR : test for 

factor VII deficiency (consider 

congenital and acquired causes such 

as liver disease, early vitamin K 

deficiency, early vitamin K 

antagonists) 

VWD Screen 

abnormal, with or 

without abnormal 

RIPA 

Quantitative or 

qualitative 

abnormality? 

Congenital or 

acquired? 

Review RIPA, 

assess multimers 

If quantitative, 

consider “low VWF” 

versus type 1 VWD 

If acquired, consider 

testing for 

hypothyroidism and a 

monoclonal 

gammopathy 

LTA abnormal 

Review pattern, potential 

causes, including VWD, 

common platelet 

disorders (e.g. secretion 

defects, dense granule 

deficiency, druginduced) 

Consider testing for a 

dense granule release 

defect and for dense 

granule deficiency if 

release is abnormal 

If Glanzmann 

thrombasthenia or 

Bernard Soulier 

syndrome is suspected, 

consider glycoprotein 

analysis, genetic testing 

If pattern or history 

suggests Quebec 

platelet disorder, 

proceed to genetic 

testing 

Hayward, Moffat, Liu. Semin Thromb Hemost. 2012;38(7):742-52 

Immediate 

bleeding? 

Check for dense 

granule release 

defects/dense 

granule deficiency if 

release is abnormal 

with all agonists 

If these tests are 

normal, consider 

conditions that may 

have been missed, 

such as Scott 

syndrome, MYH9- 

related disorder, 

etc. 

Undefined 

disorder? 

All test results normal 

Delayed bleeding? 

Mild factor 

deficiency or a 

fibrinolytic defect? 

Factor VIII and IX 

levels, others if no 

cause found 

Factor XIII levels 

α2-plasmin inhibitor 


time (short if severe 

PAI-1 deficiency) 

Genetic test for 

Quebec platelet 

disorder 

Factor VIII (two 

stage if available) 

and other factor 

assays to exclude a 

mild factor 

deficiency that 

might have been 

missed by 

coagulation 

screening tests

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