Hemostasis testing at Barnes

Jewish Hospital Uncovered

Charles Eby MD
August 7, 20
 outline
• Screening coagulation tests
• Testing for suspected bleeding disorder
– Coagulopathy
– Primary hemostasis
• Thrombophilia testing
• Monitoring antithrombotic therapy
– Anticoagulation
– Antiplatelet
aPTT

PT

TT
 Screening coagulation tests
aPTT: 21-36.5 s.
PT: 11-15 s.
TT: 18-22 s.
STAGO
Performed on undiluted plasma
Diagnostica
Sensitive to pre-analytical variables
Reference ranges:
Mean +/- 2SD of a
healthy population Automated coagulation line

2.5% of healthy people

will have prolonged results
Tolerate minor prolongations in
patients without bleeding risks

Once uncapped, stability is limited to a few hours for most analytes

 Why are underfilled blue top tubes
rejected?
• Blue top tubes contain 0.5ml NaCitrate, and
are designed to fill with 4.5 ml of blood.
• Underfilling tubes increases citrate:plasma
ratio
• Can prolong aPTT>PT
• Tolerance: 90% based on height of blood
column
 Thrombin time and fibrinogen
• TT • Fibrinogen
• Extremely sensitive to • Ref. interval: 170-
heparin and DTI 440mg/dl
• prolonged TT repeated • Method: thrombin added
after adding protamine to diluted plasma,
(TTc) monitor time to clot
• Prolonged TTc: • Calibrated against a
– DTI standard of purified
– Hypofibrinogenemia fibrinogen to convert to
– dysfibrinogenemia mg/dl
• Lower limit of detection:
60mg/dl
 PT
• One stage clotting test
• STAGO rabbit brain thromboplastin + Ca++
• ISI = 1.26
• INR=( PT ratio)ISI : (25/12.5)1.27= 2.4
• Contains heparin neutralizing material
• Hospital labs do not perform
POC/fingerstick PT/INRs
 aPTT
• 2 step clotting test:
– 1st: add activator and
phospholipid-wait
– 2nd: add Ca++
– Wait for clot to form
• Insensitive to LA
• Sensitivity to:
– FVIII ~ 30%
– FIX ~ 35%
50:50 Mixing studies for prolonged PT
or aPTT
• Only PT prolonged • aPTT only
• Why bother? • Minor prolongations
• Specific inhibitors? rare (37~40 s.)will correct
• LA only prolonging PT? • Definition of correct?
also rare – within ref. interval: too
stringent
• Clinical condition will
– Don’t be too rigid
explain multiple factor
deficiencies • Immediate and 60 min
incubation: differentiate
non-specific from FVIII
inhibitor
 Two different 50:50 mix stories
• PT 16.4 s aPTT 89 s • PT 13.9 s aPTT 56 s
• TT 11.5 s • TT 20.5 s
• 50:50 mix • 50:50 mix
• Immediate: 52 s • Immediate: 49 s
• 60O: 72 s • 60O: 48 s
• FVIII: <1% • FVIII and FIX “inhibitor
• FIX, XI ‘inhibitor patterns”
patterns”
 Coagulation factor assays
25% FVIII
Activity, no
inhibitor
pattern

25% FVIII
Activity,
Inhibitor pattern
 Factor inhibitor patterns
FVIII inhibitor Lupus Anticoagulant
FVIII FIX FXI FVIII FIX FXI
1:5 <1 42 33 1:5 20 24 ND
1:10 1 65 49 1:10 36 40
1:20 2 91 67 1:20 59 64
1:40 2 112 68 1:40 86 88
Report FVIII 1%, FIX 112%, FXI report activities for highest
68%, “inhibitor pattern dilution with comment:
present” inhibitor pattern present”
 Coagulopathy work ups
47 male, bled with tooth ext at Eldery woman, metastatic renal
37. Occupation: lumberjack cell CA, spont hematomas
• aPTT 57 s PT nl • aPTT 141
• 50:50 mix 34 s/35 s • 50:50 mix 63 s/125 s
• FVIII: 391% • FVIII 1%
• • FIX: 84%, inhibitor pattern
FIX: 3%
• FVIII inhibitor titer:
• 88 Bethesda units
• Defn: reciprocal of plasma
dilution that neutralizes
50% of FVIII in normal
plasma after 2 hr incubation
 Continued:
4 year old with epistaxis 47 woman, MVA, hip fx
• aPTT 134 s PT normal • PT 25 s aPTT nl
• 50:50 mix • Mixing study not done
– Immediate: 34 s
• FVII
– STAGO PT reagent: 5%
– 60 min: 34 s
– Innovin PT reagent 31%
• Severity of FVII deficiency
• FVII, FIX, FXI nl dependent upon species of
• FXII<1% thromboplastin: FVII Padua
 Suspected primary hemostasis
disorders
• Platelet count adequate and smear reviewed
• Global screening test for primary hemostasis:
– Bleeding time discontinued 2007
– PFA-100 substituted
 Utility of PFA-100
• Sensitive for: • Prolonged closure times
– Aspirin inhibition of cyclo- caused by:
oxygenase • Hematocrit < 30%
– Severe congenital qualitative
platelet disorders • Platelet < 100,000
• Glansmann • Other medications?
• Bernard Soulier – Antibiotics
– Type 3 vWD – SSRI
• Moderate sensitivity: – Ca channel blockers
– Type 1 vWD – Nitrates
– Plavix inhibition P2Y12 – Etc
– Mild congenital platelet • Not validated for predicting
defects
pre or post-op bleeding
Special Hemostasis Testing Area
(next to flow cytometry)
Platelet induced aggregation studies
• Typical agonists:
• ADP, collagen,
arachadonic acid
epinephrine, ristocetin
• Patient requirements:
– High suspicion for
congenital plt disorder
– Out-patient
– Minimal medications
– Nl plt count
• Takes 40 ml blood, 4 hrs
 Von Willebrand Dz work up
• In house tests: • Send out tests:
• aPTT, FVIII • vWF multimer analysis
• PFA-100 – Not part of initial workup
• – Indicated for suspected type
vWF antigen (Mon + Thurs) 2A/2B/2M, type 3
• vWF “activity” ristocetin • Factor VIII binding assay
cofactor assay (Mon – ELISA test for possible type
+Thurs) 2N vWD (defective binding of
• If type 2A or 2B suspected: FVIII to vWF)
Ristocetin induced platelet • Collagen binding assay
aggregation (RIPA) – ELISA method
– Sensitive for type 1 and type
2A/2B vWD
– Not popular in US
 Thrombophilia workup

• Be discerning in whom you work up

• Testing often done at the wrong time:
– Acutely ill
– Heparin/warfarin started
– Pregnant or on OCPs
• How will the results affect management?
 Antithrombin deficiency
Measurement:
Chromogenic activity assay:
1. AT (?) + heparin + FIIa
• Directly inhibits =incubate
thrombin and FXa 2. Residual FIIa hydrolyzes
• Prevalence ~ 1/2000 substrate
3. Change in OD inversely
• Deficiency classification proportional
– Type 1: quantitative to antithrombin activity
– Type 2: qualitative Reference interval: 80-120%
• Reactive site defect Inherited deficiency: <50%
• Heparin binding defect Acquired deficiencies: heparin,
• pleotropic dilution,
Liver dz, DIC, L-asparaginase,
nephrotic syn
Protein C and S deficiencies
• Indirectly regulate IIa
• Vitamin K dependent
• Prevalence:
– Protein C def. ~ 1/300
– Protein C def. ~1/800-
• Reference intervals:
– Protein C activity
– Free protein S antigen
• Overlaps with
“deficient” patients
 Protein C and S deficiencies
• Protein C • Protein S
– Type 1 quantitative – Type 1 quantitative
– Type 2 qualitative – Type 2 qualitative (rare)
• Test methods – Type 3 strange
– Activity clot based • Nl total antigen, decreased
free antigen and activity
– Activity chromogenic • Test methods
– 80-160% – Activity clot based
– Antigen concentration – Free protein S antigen
– 58-169%
– Total protein S antigen
 Alternate causes of C and S
deficiencies
• Protein C • Protein S
• Sick patients • Same factors and:
• Liver dz • Estrogens
• Vit K deficiency – OCPs, 2nd/3rd trimester

• Warfarin • Test method dependent:

– Protein S activity assays
• DIC
– Risk of false positive due to
FVL het/homozygosity
 Activated protein C resistance
• Laboratory phenotype
of FVL
• Very sensitive screening
test
• Cut-off ratio of 1.7
• Performed Mon + Thurs
• If abnormal, reflexed to
FVL genotype
 FVL and prothrombin G20210A
• Genotyping performed in BJH molecular
diagnostic laboratory
• TAT- 1-2 weeks
 hyperhomocysteinemia

MTHFR 677T>C mutation

TT CT CC
65% 25% 10%

Testing available at BJH

 Fibrinolysis pathway
• Only in-house test:
• Quantitative D-dimer
• Method:
immunoturbidity
• Latex particles coated
with anti D-D agglutinate
increase light
transmission
• STAGO test not FDA
approved to rule out VTE
in low-moderate risk
patients
 Lupus Anticoagulant testing
• ISTH guidelines (revised 2009)
• Patient selection:
• Low: elderly (>60)
• Moderate (recurrent preg loss, provoked VTE,
elevated aPTT)
• High: unprovoked VTE, CVA,CAD < 50,
thrombosis in unusual place
 LA testing
• OK if heparin activity < .8U/ml
• OK if INR <3.0
• Reference intervals 3SD
• Express results as ratio of normal plasma tested
concurrently
• Two sensitive screening methods
– dRVVT
– aPTT
• Provide a written interpretive report
 Anticoagulation therapy monitoring
• Heparin
– aPTT- printed nomogram
– Correlated to anti-Xa activity
• Heparin and LMWH
– Hybrid calibration curve for anti-Xa activity
• Foundaparinux
– No demand, no assay
• Argatroban/bivalirudin
– aPTT-printed dosing guidelines
– Prolong PT/INR as well
 Antiplatelet therapy monitoring
• Asprin and plavix “resistance”
• PFA-100- sensitive for aspirin inhibition but
not plavix
• Modified platelet aggregation study
– ADP 5mM and arachadonic acid
– “Sensitive” if ADP aggregation <40%, AA <20%
• Coming soon: Verify Now
 Verify Now

Cartridges for platelet inhibition by aspirin, plavix, and Reopro

Output in arbitary units: ARU, PRU
Cut-offs based on limited clinical data
 HIT testing
• In house: PF4-heparin antibody detection by
ELISA (STAGO)
• Noon cut-off M-F
• Weekend by LMR review: cut-off noon
• Results: positive or negative based on OD cut-off
(0.5 OD +/-10%)
• Growing consensus that specificity improved
without sacrificing sensitivity when using cut-off
of > 1.0 OD
• Lab will start reporting OD soon
 HIT testing
• Functional assays
• Seratonin release assay
– SLUH
– Tues and Fri
– Sample must arrive by noon
• Plasmas for PF4-heparin ELISA are retained,
can be sent out upon request