1st and 2nd hemostasis

Question 1

Function of Risticetin

Answer 1

Action depends on vWF and GP1b/IX/V

Question 2

vWD

Answer 2

Corrected result after adding normal plasma

Question 3

Bernard-Soulier syndrome

Answer 3

Genetic GP1b/IX receptor deficiency

Not corrected after adding normal plasma

Question 4

PFA-100 specimen requirement

Answer 4

800-900 uL blood or PRP in citrate tube
PRP must be undisturbed in 30min
Whole blood: dilute 1:10 with saline and tested within 4 hrs

Question 5

PFA-100 result reporting

Answer 5

PLT count, acitivity, vWF, hematocrit

Question 6

PFA-100 vs Aspirin use

Answer 6

Col/Epi: Abnormal in Aspirin use, VWD, Glanz-Mann

Co/ADP: Normal in Aspirin use. Abnormal in VWD, Glanz-mann

Question 7

PLT aggregation wave analysis for normal pt

Answer 7

ADP, Epinephrine, Ristocetin: Biphasic curve

Collagen, Arachidonic acid: Monophasic curve (only 2nd wave)

Question 8

Arachidonic acid and U46

Answer 8

Screening for pt using aspirin last 7-10 days
U46 binds to TXA2 receptor & not affected by aspirin
- AA abnormal + U46 normal: just aspirin
- AA abnormal + U46 abnormal: receptor defect

Question 9

Primary vs Secondary aggregation

Answer 9

Primary: Response to agonist, shaped change and small aggregation, reversible

Secondary: Response to endogenous ADP, complete aggregation, irreversible

Question 10

Glanzmann’s thrombasthenia

Answer 10

GPIIb/IIIa deficiency —> Can’t bind to fibrinogen to make a clot —> No response to any aggregation factor, except Ristocetin

Question 11

Prothrombin time (PT) sample requirement

Answer 11

• Citrated patient’s PPP or control PPP

Question 12

PT: key reagent - purpose - procedure

Answer 12

• Thromboplastin: TF (from brain, lung, placenta) + Pl + CaCl2
• Screening extrinsic and oral anticoagulant
• Add reagent to PPP and record the time for fibrin formation

Question 13

PTT: key reagent - purpose - procedure

Answer 13

• Activated partial thromboplastin: small amount of Pl (not activate F7) + surface activator (silica, kaolin) + CaCl2
• Screening intrinsic, inhibitor (Heparin affect APTT)
• Add APTT reagent, add Ca, record the time for fibrin formation

Question 14

PT abnormal

Answer 14

• Prolonged:
  + Deficiency in VII, X, V, II, fibrinogen
  + Inhibitor
  + Dysfunctional vitamin K due to Warfarin/Coumadin
• Shortened: Pt is on recombinant VIIa therapy

Question 15

PTT abnormal

Answer 15

Factor deficiency (acquired, inherited)
A present of inhibitor: Lupus, FVIII inhibitor
Heparin

Question 16

FI (Kinetic fibrinogen)

Answer 16

• Quantitative test: low conc. of fibrinogen (diluted plasma) + high thrombin
• Make 1:10 dilution for a standard curve. Make several dilutions –> add thrombin –> record clotting time.

Question 17

FI abnormal

Answer 17

High: inflammation, pregnant, oral contraceptive
Low: DIC, fibrinolysis, liver disease (fibrinogen made in liver)

Question 18

TT (Thrombin Clotting Time)

Answer 18

• Qualitative test: high conc of fibrinogen (undiluted plasma) + low thrombin
• Add thrombin –> record clotting time

Question 19

TT abnormal

Answer 19

• Prolonged:
  + Heparin (antithrombin) or Direct thrombin inhibitor
  + Hypofibrinogen, dysfibrinogen
  + Elevated Fibrin degradation products
• Shortened
  + Inappropriate specimen
  + Protamine sulfate infusion (used for patient after a long term heparin therapy)

Question 20

Correction the dilutions for FI standard curve

Answer 20

• Clotting time > 50s: too little fibrinogen –> Make 1:5 instead of 1:10 dilution
• Clotting time < 5s: too much fibrinogen –> Make 1:20, 1:40 –> Multiple with dilution factor

Question 21

International Normalized Ratio (INR)

Answer 21

• Range: 0.8-1.3
• Purpose: address the high level of variability in reagent sensitivity; standardize reports
• INR = R^ISI
  + R: patient’s PT/mean normal PT
  + ISI: Int’l Sensitivity Index

Question 22

Screening F13

Answer 22

• Purpose: Deficiency of F13 makes fibrin more soluble as a lack of cross-linking
• Procedure: Patient’s plasma + CaCl –> clot: keep 37C in 1hr –> Add 5-6M urea (break H-bond)
• Result: Clot dissolves within 24hrs –> Deficiency of F13 (< 1-2%)

Question 23

Mixing test

Answer 23

• Purpose: Differentiate factor deficiency vs inhibitor’s presence
• Procedure: Mixing pt’s plasma with normal plasma and repeat tests (PT, APTT)
  + Corrected result: Factor deficiency (compensated from normal plasma)
  + Uncorrected result: Inhibitor’s presence (inhibit the normal plasma)

Question 24

Specific Coagulation Factor Test

Answer 24

• Purpose: Confirm the specific factor deficiency, understand the activity of a factor
• Principle: Add diluted pt’s plasma to a known-factor deficient plasma and check if it can correct the prolong PT/APTT time

Question 25

Reptilase time

Answer 25

- Purpose: Differentiate between dysfibrinogenemia and FDP's presence - Principle: Venom act as serine protease --> cleave fibrinogen to fibrinopeptide A (instead of A&B from thrombin) - Reptilase is insensitive to heparin - Result: + Reptilase time > TT: Dysfibrinogenemia + Reptilase time < TT: Presence of fibrin degradation products + If Reptilase time is normal (TT: prolonged): Heparin contamination

Question 26

Diluted Russell's viper venom test

Answer 26

- Purpose: Activate F10 (similar method to the PT) to look for phospholipid Abs (inhibitor) - Procedure: Diluted venom + Pl + Ca --> Activated F10 --> Record clotting time - Result: prolonged --> Mixing test to R/O factor deficiency --> Add extra phospholipid: If correct, prolonged due to LA/aPL

Question 27

Kaolin Clotting time

Answer 27

- Principle: Most sensitive test. Detects all class of inhibitors, include VIII inhibitor - Similar to APTT but w/o added phospholipid (rely on residual cell fragment and plasma lipids to provide phospholipid surface) - Kaolin: Negatively charged activator --> incubated with test plasma --> activate contact factors of the intrinsic pathway.

Question 28

Platelet Neutralization Procedure

Answer 28

- Principle: Look for anti-phospholipid Abs by adding extra phospholipid from ruptured PLT - Procedure: Add pt's PPP with ruptured PLT --> Add PTT reagent + Ca --> Record clotting time --> Compare pt's PPP + saline vs pt's PPP + ruptured PLT. - Result: Ruptured PLT mix < Saline mix --> Presence of aPL Abs

Question 29

Bethesda Titer Assay

Answer 29

- Specific Factor Inhibitor Assay (not LA/aPL) - Measure F8 inhibitor - 1 Bethesda unit = amount of inhibitor needed to neutralize 50% of 1 unit F8:C in normal plasma at 37C

Question 30

Bethesda Procedure

Answer 30

- Dilute patient's plasma 1:2 - 1:1024 and mix with equal volume normal plasma for 2hrs at 37C - Find dilution with residual F8:C closet to 50% (30-60%)