Hematology: Coagulation Lab Evaluation

Front 1

Screening tests

Back 1

- Primary hemostasis: platelet count &/or bleeding time
- Secondary hemostasis: PT & aPTT
- Sometimes used to assess hemostatic status prior surgery.

Front 2

Specimen requirements

Back 2

- Plasma from a whole blood specimen
- Mixed w/ 9:1 (blood:anticoagulant)
- 3.2% solution of sodium citrate (binds to Ca+ to prevent coagulation).
- order of draw: first or immediatel after non-aditive tube.

Front 3

Unacceptable specimen/rules

Back 3

- "Short draw": false prolonged result due to excess anticoagulant.
- Clotted specimen
- Excessive specimen agitation--> hemolysis, procoagulant & platelet activation.
- Visually hemolized specimen.
- Excessive manipulation of needle during phlebotomy--> release of procoagulant substances (falsely shortened results).
- Lipemic/icteric plasma: false results usin optical clot based testing.
- Slowed/stopped venous circulation--> falsely decreased results
- "Flush" & discard 5ml of blood when collecting from central or peripheral line.

Front 4

High hematocrits (>55%)

Back 4

- The 1:10 ratio is effective provided that the hematocrit is 55% or less.
- Hematocrit >55%, decrease in plasma volume--> falsely prolonged clot based coagulation test results.

Front 5

Hemostasis specimen storage temperature

Back 5

- Plt. aggregometry: never refrigerate sample (18-24 Celsius)
- PT: 18-24 Celsius
- PTT: 18-24 Celsius (never warm- heat destroys factor 8 & 5).

Front 6

Hemostasis specimen storage time

Back 6

- Plt. aggregation: w/in 3 hrs of collection.
- PT: test w/in 24 hrs of collection.
- PTT (no heparin): test w/in 4 hrs of collection.
- PTT (heparin therapy): centrifuge w/in 1 hr & remove plasma. Test w/in 4 hrs.
- Long term hemostasis storage: -20 Celsius for 2 wks.; -70 Celcius for 6 mths.
- Rapid thaw at 37 Celsius.

Front 7

Hemostasis specimen preparation

Back 7

- Plt. aggregation: requires plt rich plasma (200-300 X 10^3/uL).
- Most clot based hemostasis testing require PPP (<10 X10^3/uL).
- PPP: plts secrete fibrinogen, factors 5, 8, & vWF--> may shorten clot based coagulation assays.

Front 8

Platelet function tests

Back 8

- Pre-test: perform plt count; review blood smear.
- Thrombocytopenia: common cause of hemmorhage.
- Abnormal plts: easy bruising, petechiae, purpura, epistaxis, & plt count >50 X 10^3/uL.

Front 9

Bleeding Time Tests

Back 9

- Screening for primary hemostasis defects.
- In vivo measurement of plt. function
- Not prolonged in most d/o of secondary hemostasis.
- Ref. range: 2-9 mins.
- Prolonged BT: thrombocytopenia, vWD, afbrinogenemia, vascualr d/o, & aspirin therapy.
- Not reliable test: poor predictive value & technique

Front 10

Platelet Aggregometry

Back 10

- Requires platelet rich plasma.
- Must be tested w/in 3 hrs of collectin.
- Aggregating agent added--> aggregation complete w/in 6-10 mins.
- Aggregation occurs in 1 or 2 waves depending on the agent used.

Front 11

Aggregometry: primary wave

Back 11

- Direct response of the plts. to the aggregation agent.
- Represents plt shape change & formation of small aggregates.

Front 12

Aggregometry: secondary wave

Back 12

Complete aggregation due to
ADP release from the plt dense bodies/delta granules.

Front 13

Platelet Aggregation activating agents

Back 13

- Thrombin, Adenosine diphosphate (ADP)- Epinephrine, collagen, arachidonic acid.
- Ristocetin: normal result indicates that vWF is present w/a functional vWF receptor (GPIb.V/IX).

Front 14

Platelet function add'l tests

Back 14

- platelet secretion studies: serotonin & ADP release
- Flow cytometry: monoclonal antibodies to plt receptors
* Bernard-Soulier syndrome (CD42): GPIb/IX/V deficient
* Glanzmann thrombasthenia (CD41, CD61): GPIIb/IIIa deficient

Front 15

Clot formation principle

Back 15

- The time interval from the initiation od coagulation to visible clot formation reflects the condition of the coagulation mechanism.
- Prolonged clotting time= coagulation deficiency.

Front 16

Clot based screening assays

Back 16

- Prothrombin Time (PT)
- Partial Thromboplastin Time (PTT)
- Thrombin Clotting Time (TCT).

Front 17

Prothrombin Time (PT)

Back 17

- Reagent: thromboplastin prepared from tissue factor suspended in PL.
- Used most often to monitor Anticoagulant Warfarin/Coumadin.
- QC: normal & prolonged specimens run every shift & each rgt change.
- PT ref. range: varies from site to site; generally= 10-13 seconds
- DIC, liver dz, Vit. K deficiency--> affect factor VII--> prolonged PT

Front 18

International Normalized Ratio (INR)

Back 18

- Used by most laboratories due to ariations among thromboplastin rgts.
- Should only be used for stable anticoagulated pts.
- INR= (PT of pt/Mean PT of normal range)^ISI

Front 19

Vitamin K vs. liver dz

Back 19

- Perform factor assay activity for factors V & VII.
- Vitamin K deficiency: factor VII conc'n
- Liver dz: factor V conc'n

Front 20

Limitations of PT

Back 20

- QNS
- Clotted
- Hemolysis, lipemic, icteric
- Hematocrit >55%
- Want rgt insensitive to heparin
- Incorrect reconstitution of rgt.
- Incorrect instrument calibration

Front 21

Partial Thromboplastin Time (PTT)

Back 21

- Reagent: contains PL & a negatively charged activator- kaolin, ellagic acid, celite
- Performed to monitor the effects of unfractioned heaprin therapy & to detect circulating anticoagulants (antibodies).
- QC: normal & prolonged specimens run every shift & w/ each rgt. change.
- Ref. range: varies from site to site- type of rgt, instrument, & pt. population.
- Often prolonged when factor levels are <30% of fibrinogen is <100 mg/dL.

Front 22

Prolonged PTT + hemorrhage

Back 22

Deficiency in factor XI, VIII, X, V, prothrombin, fibrinogen.

Front 23

Prolonged PTT w/o bleeding

Back 23

Deficiency in fatcor XII, prekallikrein, or high molecular weight kininogen.

Front 24

Prolonged PTT

Back 24

- Presence of specific inhibitor: anti-factor VIII or IX.
- Presence of non- specific inhibitor: LA, ACLA
- Presence of interfering substances- fibrin degradation products or paraprotein

Front 25

Acquired intrinsic deficiencies

Back 25

- DIC: procoagulant consumptions
- Vit. K deficiency: not as sensitive as PT but PTT eventually prolonged (II, VII, IX, X)

Front 26

PTT mixing studies

Back 26

- Used for LA & factor inhibitors.

Front 27

Thrombin Clotting Time (TCT or TT)

Back 27

- Commercially prepared bovine thrombin rgts cleaves fibrinopeptide A & B from plasma fibrinogen (E doamin) to form a detectablefibrin polymer.
- QC: normal & prolonged specimens run each shift & w/ each rgt change.
- Ref. range: 15-20 secs.

Front 28

Prolonged TCT/TT

Back 28

- <100 mg/dL fibrinogen (hypofibrinogen)
- Presence of anti-thrombotic materials, i.e. FDPs, paaraprotein, heparin
- Afibrinogenemia
- Dysfibrinogenemia

Front 29

Reptilase Time (RT)

Back 29

- Reptilase (venom of Bothrops atrox): directly catalyzes the conversion of fibrinogen--> fibrin; insensitive to the effects of heparin.
- Cleaves only fibrinopeptide A not B.

Front 30

Prolonged RT

Back 30

- Indicates decreased functional fibrinogen (dysfibrinogenemia or hypofibrinogenemia)
- Also seen in the presence of FDPs & paraproteins.

Front 31

Fibrinogen assay

Back 31

- Recommends clot based method of Clauss to estimate the functional fibrinogen level.
- Thrombin rgt is 25X more concentrated that TCT assay.
- PPP is diluted 1:10
- Inverse, linear relatonship between time to clot & conc'n of functional fibringen
- Ref curve is prepared using reference plasma (fibrinogen levels; factor I assay).
- Results of fibrinogen assay are compared to results of the ref. plasma
QC: normal & prolonged specimens are run each shift & w/ each rgt. change.
- Ref. range: 200-400 mg/dL

Front 32

Hypofibrinogenomia

Back 32

Associated w/ DIC & severe liver dz.

Front 33

Increased fibrinogen

Back 33

May caused by moderately severe liver dz, pregnancy, chronic inflammatory condition.

Front 34

Single factor deficiency

Back 34

- PTT= prolonged (w/ no ready explanation, i.e. heparin therapy, LA, factor specific inhibitor).
- PT & TCT= normal
- Factor 8 (hemophilia A), 9 (hemophilia B), or 11 (hemophilia C)
- PT & PTT= prolonged (w/ no ready explanation, i.e liver dz, DIC, oral anticoagulant therapy, Vit. K deficiency)
- TCT= normal
- Factor 2 (prothrombin), 5, or 10 deficiency.
- Factor 7 deficiency: PT= prolonged; all other tests are normal.

Front 35

Factor VIII assay

Back 35

- Use PTT to estimate the conc'n of functional factor 8 by incorporating prepared factor 8 depleted/deficient plasma--> provides normal activity of all procoagulants EXCEPT factor 8.
- Ref. range: 50-150%
QC: normal & prolonged specimens are run during each shift & w/ each rgt change.

Front 36

Factor XIII

Back 36

- Strengthens the fibrin clot & renders it resistant to proteases.
- Final event in plasma coagulation & is essential for normal hemostasis & wound healing.
- levels <5%--> hemorrhage
- Decreased in congenital factor 13 deficiency, acquired DIC, secondary to Crohn's dz, leukemias, ulcerative colitis, sepsis, inflammatory bowel dz, surgery.

Front 37

Factor XIII deficiency

Back 37

- Infants: bleeding is evident w/ seepage at the umbilical stump.
- Adults: slow/progressive bleeding w/ poor wound healing & slow resolving hematomas.
- Pt. presents delayed bleeding & poor wound healing; PT, PTT, platelet count, & fibrinogen level are normal

Front 38

Factor XIII test

Back 38

- 5M urea solubility test.
- Unstable clot dissolves in 5M urea solution--> intact for at least 24 hrs in urea.

Front 39

Fibrinolysis Tests

Back 39

- Quantitative D-dimer immunoassay
- Plasminogen substrate assay
- Tissue plasminogen activator (TPA) assay
- Plasminogen activator inhibitor-1 (PAI-1)

Front 40

Quantitative D-dimer Assay

Back 40

- During coagulation, fibrin polymers cross linked/stabilized by factor 13 & bind plasma & TPA.
- TPA acivates plasminogen--> plasmin.
- Bound plasmin cleaves fibrin--> FDPs & D-dimers
- Convinient for detectng reactive fibrinolysis.
- Indirect implication of thrombosis.

Front 41

Fibrin Degradation Products (FDPs)

Back 41

- Largely replaced by the D-dimer assay
- Semi-quantitative
- Increased FDPs: DIC, DVT, PE, & systemic fibrinolysis(liver dz or after thrombolytic therapy)

Front 42

Plasminogen Substrate Assay

Back 42

- Normal plasminogen activity: 5-13.5mg/dL
- TPA/Urokinase converts plasminogen bound to fibrin--> plasmin
- Bound plasmin degrades fibrin.
- Free plasmin rapidly inactivated by alpha2 antiplasmin.
- Decreased: thrombolytic therapy, DIC, hepatitis, cancer, hereditary deficiencies (promyelocytic leukemia).
Increased: Inflammation, pregnancy hemorrhage), systemic fibrinolysis.

Front 43

Plasmin Substrate Assay: chromogenic assay

Back 43

- Streptokinase (from beta streptococcus) added to pt. PPP--> binds to & activates plasminogen--> streptokinase/plasmin mixture reacts w/ chromogenic substrate--> yellow color.
- Color intensity proportional to the original plasminogen conc'n
- Ctrl plasma is tested w/ unknown pt. PPP

Front 44

Tissue Plasminogen Activator (TPA) Assay

Back 44

- Physiological human plasminogen activators: TPA & urokinase.
- Both activators are serine proteases that dorm complexes with plasminogen at the fibrin surface--> activating plasminogen & initiating thrombus/clot degradation.
- Both are inactivated by the endothelial secretions or plt alpha granules.

Front 45

Plasminogen Activator Inhibitor-1 (PAI-1)

Back 45

- Inactivates TPA
- Increased: strongly associated w/ venous thrombosis (& maybe a cardiovascular risk factor)
- Decreased: rare; total absence--> hemorrhage
- Can be measured w/ an enzyme immunoassay or chromogenic assay (indirect: using TPA in the plasminogen; known amount of TPA is added).

Front 46

Conditions w/ excessive fibrinolytic activity

Back 46

- Inflammation & trauma- radical increase in circulating plasmin--> hemorrhage
- Bone trauma, fractures

Front 47

Fibrinolysis deficiencies

Back 47

- Occur when TPA pr plasminogen levels become depleted
- When excess secretion of PAI-1 depresses TPA activity

Front 48

Warfarin/Coumadin therapy

Back 48

- Vitamin K antagonist
- Factors 2, 7, 9, 10 & coagulation ctrl proteins C & S depend on vitamin K
- Vit. K responsible for the post-translational modification that enables factors to bind to Ca2+ & PL.
- Warfarin/Coumadin suppresses gamma-carboxylation of glutamic acid- necessary for binding site of coag factors.
- Prophylaxis: Prescribed to prevent strokes & to prevent thrombosis after trauma, or surgery.
- Therapy: Inhibits recurrence of DVT or PE & controls coagulation after acute MI
- Therapeutic levels takes about 5 days.
- Protein C depletes quickly (short half-life)--> prothrombotic (due to lack of mechanism to shutdown coag pathway).
- Pt. prothrombotic for the first 2-3 days due to loss pf protein C pathway--> Tx: warfarin w/ heparin therapy as well for first few days.

Front 49

Warfarin/Coumadin monitoring

Back 49

- PT assay: sensitive in reductions to 3 out of 4 dependent factors
- Factor 7 polong the PT w/in 24 hrs.
- Tharapeutic when factor assay levels are <50% (at least 3 days).
- Essential close monitoring: narrow therapeutic range; out of range--> thrombosis or hemorrhage.

Front 50

International Normalized Ratio (INR)= [Patient PT/Mean PT of normal range]^ISI (International Sensitivity Index)

Back 50

- Utilized due to variations among thromboplastin rgts.
- Should only be used for stable anticoagulant pts.
- Therapeutic range: 2-3 if mechanical valve heaert valve is in place.
INR>4- increased risk of hemorrhage.

Front 51

Pitfalls of Warfarin therapy

Back 51

- Dietary it. K decreases the warfarin effect & reduces the INR.
- Direct thrombin inhibitors prolong PT.
- Reversal of warfarin overdose based on teh INR & bleeding.

Front 52

Heparin

Back 52

- Activates antithrombin to neutralize serine proteases.
- Antithrombin inactivates thrombin, IXa, Xa, XIa, & XIIa
- aPTT: lab test of choice monitoring heparin therapy
- TT: most sensitive assay for the presence of heparin.

Front 53

Unfractionated Haparin (UFH) therapy

Back 53

- Administered intravenously.
- Use to treat DVT, PE, acute MI, & prevent re-occlusion after stent placement & during cardiopulmonary bypass & dialysis.
- Stopped at 5 days to avoid HIT w/ thrombosis.

Front 54

UFH monitoring

Back 54

- Perform PTT
- Baseline PTT is collected prior therapy begins.
- Prolonged PTT: Intrinsic factors; specific inhibitors- factors 8, 9, 11; Lupus coag.
- Another specimen collected 4-6hrs after initiation of therapy.
- Must monitor plt. count: 40% or > reduction is evidence of HIT.
- Must be replaced immediatey w/ more expensive DTIs.

Front 55

PTT limitations

Back 55

- Heparin resistance
- Hyperfibrinogenemia
- Factor VIII >150%
- Antithrombin depletion (normal PTT)
- Independent of heparin levels
** Reversal of UFH overdose: prtamine sulfate neutralizes UFH (as well as LMWH- incomplete neutralization).

Front 56

Low Molecular Weight Heparin (LMWH)

Back 56

- Derived from UFH using chemical fractionation
- Provides the same active sequence as UFH but provides little bridging surface due to ebing shorter--> reduced antithrombin response & HIT.
- Anti- factor Xa response is unchanged.
- Administered by subcutaneous injection (fixed dose in syringe).

Front 57

Advantages of LMWH

Back 57

- Rapid bioavailability after injection
- Half-life of 3-5hrs. (1-2 for UFH)
- Fixed dose response reduces the need for lab monitoring.
- Reduced HIT risk due to reduced bridging
- No change in bleding risk as UFH
- Can be given on outpatient basis.

Front 58

LMWH monitoring

Back 58

- Cleared by kidney
- Tested w/ chromogenic anti-fatcor Xa assay due to PTT's insensitivity to LMWH.
- Therapeutic ranges:
* 2 injections/day: 0.5-1.0 IU/mL
* 1 injection/day: 1.0-2.0 IU/mL

Front 59

DTIs

Back 59

- Agatroban
- Lepirudin
- Bivalirudin

Front 60

Agatroban (DTI)

Back 60

- Reversibly binds & inactivates free & clot bound thrombin (w/o involving AT).
- Administered intravenously & cleared by liver
- 5% bleeding risk
- Short half-life (51mins) clears from blood in 2-4hrs.

Front 61

Lepirudin (DTI)

Back 61

- Reversibly binds & inactivates free but not clot/fibrin bound thrombin (large molecule)
- Administered intravenously & cleared by kidney.
- 10% bleeding risk
- Short half-life (20mins) clears from blood rapidly

Front 62

Bivalirudin (DTI)

Back 62

- Reversibly binda & activates free & clot/fibrin bound thrombin.
- Intended for use w/ aspirin
- Administered intravenously & cleared by kidney
- 4% bleeding risk
- Short half-life (25mins) clears from blood rapidly.

Front 63

Monitoring DTIs

Back 63

- Affect TT, PT, & PTT
- Lepirudin/Bivalirudin: blood collected 4 hrs after the start of IV therapy
- Argatroban: blood collected 2 hrs after the start of IV therapy

Front 64

Anti-platelet therapy

Back 64

- Aspirin: irreversibly acetylates the plt enzyme cyclooxygenase
- Given to prevent acute MI & stroke
- 10-15% of individuals taking aspirin generate inadequate lab response as measured by plt aggregometry.