Coagulation tests

Question 1

Primary vs secondary hemostasis

Answer 1

• Hemostasis: the ability to clot
• Primary: initial platelet plug (front line defense)
• Areas of body under its control: skin, mucosa, endothelium
• Things involved: platelets, vonWillebrand’s (vW), fibrinogen
• Secondary: proteins to finalize the plus
• Areas under control: joints, deep tissue
• Involved: clotting factors

Question 2

Manifestations of hemostasis deficiencies

Answer 2

• Primary: can see from the skin (petechiae, ecchymoses, purpura), prolonged bleeding time (BT)
• Secondary: can only see in joints

Question 3

Tests of primary hemostasis

Answer 3

• History (menstrual, dental, family)
• Peripheral smear (look for abnormal platelets)
• Bleeding time (make thin slice in skin and measure time to clot, should be <9min)
• PFA100 (platelets function analyzer)

Question 4

Inherited conditions that prolong BT

Answer 4

• Glanzmann’s thrombasthenia (IIbIIIa receptor deficiency)
• Bernard Soulier (IbIX receptor deficiency)
• vW disease (vWD; defective or low vW)
• Platelet granule defects

Question 5

Acquired conditions that prolong BT

Answer 5

• Uremia
• Paraproteinemia
• Severe anemia
• Liver disease
• Drugs (NSAIDs, aspirin)
• BT does not correlate w/ exposure to NSAIDs

Question 6

PFA100

Answer 6

• Anticoagulated whole blood passed through pore coated w/ collagen + ADP
• Measure closure time (CT), time it takes to occlude aperture via clot
• Superior test to BT as a screen for platelet dysfunction (can detect moderate to severe vWD, aspirin’s effect)
• Cannot distinguish btwn type 1 vWD (not enough vW) and type 2 vWD (poor quality vWD)

Question 7

Specific tests of platelet function

Answer 7

• Platelet aggregometry: tests platelet response to agonists that induce aggregation (rare diseases)
• vWF Ag: quantitaive measure of vWF (vW made in endothelial cells, amount in blood surrogate to amount being made)
• Ristocetin cofactor assay (RCA): tests the ability of vWF to bind to its receptor (GPIbIX)
• RCA tests the functional aspect of vW (the high weight multimer form)
• If vWF Ag and RCA do not parallel, usually means problem in activity (quality) of vW
• Comparing the vWF Ag and RCA allows you to distinguish btwn type 1 and type 2 vWD (both low-> type1, vWF Ag normal and RCA low-> type2A or M)

Question 8

Secondary hemostasis (coagulation cascade) 1

Answer 8

• 3 parts: instrinsic, extrinsic, and common pathways
• Extrinsic: starts w/ tissue factor (TF) activating factor VII to its active form VIIa (requires Ca and phospholipid, PL)
• VIIa+TF+Ca/PL is able to activate factor X to Xa

Question 9

Secondary hemostasis (coagulation cascade) 2

Answer 9

• Common: starts w/ X being activated to Xa (requires Ca and PL), either by TF+VIIa or IXa+VIIIa complex (from extrinsic pathway)
• Once Xa is activated, it complexes w/ active Va (activated by trace thrombin) and Ca/PL (Xa/Va/Ca/PL is prothrombinase complex) to activate prothrombin (factor II) to thrombin (IIa)
• Once thrombin is active it cleaves fibrinogen (factor I) to fibrin (Ia), which is insoluble and precipitates out of solution to form the clot

Question 10

Secondary hemostasis (coagulation cascade) 3

Answer 10

• Intrinsic: Starts w/ factor XIIa (activated by negatively charged surfaces) which complexes w/ HMWK (high molecular weight kininogen), PK (prekallekrein), and factor XI to activate XI-> XIa (XI usually activated by thrombin)
• XIa activates factor IX (w/ Ca) to IXa (IX can also be activated by VIIa+TF)
• IXa must complex w/ active VIIIa (activated by trace thrombin) and Ca to form the intrinsic tenase complex
• This IXa/VIIIa/Ca/PL tense complex activates X to Xa and thus begins the common pathway

Question 11

-Tests for secondary hemostasis

Answer 11

• PT (prothrombin time): tests the extrinsic + common pathways
• aPTT (activate partial thromboplastin time): tests the intrinsic + common pathways

Question 12

aPTT

Answer 12

• Add citrated (anticoaged) plasma + PL + contact activator + Ca and measure time to clot
• Most sensitive to the intrinsic pathway (prolongs when 60-70% deficient)
• Only test that is sensitive to heparin (PT test the system is overloaded w/ TF and is not sensitive to heparin)
• More sensitive to VIII and XI than to IX
• Less sensitive to thrombin and fibrinogen
• Accuracy depends on reagents, mild but significant deficiencies can be missed, deficiencies in common pathway prolong both aPTT and PT
• Correlation btwn degree of prolongation and factor level better for VIII than others
• HMWK, prekallikrein, and XII affect the aPTT but deficiencies in these do not cause bleeding
• Normal aPTT in adult: 23-33

Question 13

Spurious prolongation of aPTT

Answer 13

• Elevated HCT (increases the citrate:plasma ratio, removing some Ca)
• Heparin in tube (inactivates thrombin and Xa)
• Clotted sample (pulls factors out of solution)

Question 14

Workup of a prolonged aPTT

Answer 14

• If an aPTT is prolonged it means there is either a deficiency in a factor or an inhibitor of a factor present, but doesn’t tell you which
• Must do a 50/50 mix (50% patient blood and 50% normal blood) to increase all factor levels to at least 50% (and therefore they are all at high enough level to clot)
• If the mix clots, that means there was a deficiency in the patients blood
• If the mix doesn’t clot, that means there was an inhibitor in the patients blood that is still inhibiting the factor(s) in the mix

Question 15

Workup based on result of 50/50 (after prolonged aPTT)

Answer 15

• If the 50/50 corrected the blood, assay for individual factors (XI, IX, VIII) to see where the deficiency lies (since XII is unimportant, but the rest are)
• If the 50/50 did not correct the blood, test for lupus anticoagulant Abs, anticardiolipin Abs
• If those Ab tests are negative then test for specific inhibitor Abs

Question 16

Prothrombin time (PT)

Answer 16

• Add citrated plasma + tissue (for TF/PL) + Ca and measure time to clot
• Measured in INR, normal being >1.3
• Measures deficiency of VII and common pathway factors
• PT begins to prolong w/ 50-60% factor deficiency
• Most common cause of isolated prolongation of PT is heterozygous deficiency of VII or gene polymorphism

Question 17

Workup of prolonged PT

Answer 17

• Also do 50/50, results indicate the same
• If 50/50 corrects the PT then there was a deficiency
• If 50/50 does not correct the PT then there was an inhibitor
• Further workup for deficiency: test for factor deficiency (usually VII)
• Further workup for inhibitor: test for thrombin Ab, other Abs

Question 18

Thrombin time (TT)

Answer 18

• Add thrombin + citrated plasma and measure clotting time
• Only measures amount and quality of fibrinogen (catalyzes the last step only)
• Minimal hemostatic level of fibrinogen: 75-100 mg/dl (fibrinogen <100 mg/dl prolongs TT)
• Abnormal fibrinogen quality seen in hereditary dysfibrogenemia, cirrhosis, HCC (?), newborns
• TT can also be prolonged in: heparin Rx, excess fibrinogen degradation products (FDPs), paraproteinemia (large proteins inhibit polymerization of fibrin), renal failure

Question 19

Hemophilias

Answer 19

• Inherited diseases, X linked recessive
• Hemophilia A (more common, less severe): deficiency in factor VIII
• Hemophilia B (less common, more severe): deficiency in factor IX
• Hemophilia C (very rare, very severe): deficiency in factor XI. NOT X linked, it is autosomal recessive, but heterozygotes can also show prolonged bleeding

Question 20

Fibrinolysis

Answer 20

• Complicated pathway to break down clots and control coagulation
• Key mediator of clot breakdown: plasmin
• Plasmin destroys fibrin polymers and breaks them up into fibrin degradation products, thus un-clotting blood

Question 21

Tests for accelerated fibrinolysis

Answer 21

• Euglobulin lysis time (ELT): measures the time to breakdown clot in a sample of plasma
• Dilute whole blood clot lysis time (DWBCLT): measures the time to breakdown clot in a sample of whole blood
• These are shortened in: advanced cirrhosis, alpha2-antiplasmin deficiency, plasminogen activator inhibitor-1 deficiency (PAI-1), systemic fibrinolysis
• Can occasionally see prolonged in venous thrombosis and renal failure (increased PAI-1 levels)