Laboratory Evaluation of Hemostasis -  For Prothrombin testing: RT and test within 24

Chapter 45 hours
 For Partial thromboplastin time: RT and test
Hemostasis Specimen Collection within 4 hours
 Most specimen: venous blood collected by
venipuncture and mixed 9:1 w/ a 3.2% solution Prothrombin Time
of sodium citrate anticoagulant
Principle
 For platelet function testing – maintained as
 PT reagents: called thromboplastin or tissue
well-mixed whole blood
thromboplastin
 Reagent is prepared from affinity – purified tissue
Patient Management During Hemostasis Specimen factor suspended in phospholipids mixed with
Collection buffered 0.025mol/L calcium chloride
 No fasting is required  Reagent + citrated plasma = fibrin polymerization
 Drugs that affect the outcomes of coagulation (activating plasma factor VII)
test:  Calcium = precipitate in + of TF:FactorVIIa
- Aspirin: suppresses most platelet complex, FactorVIIIa:FactorIXa complex and
FactorVa:FactorXa complex
function
 + clot formation
- Warfarin: reduces the activities of
factor II, factor VII, factor IX, factor X,
Prothrombin Time
protein C, protein S and protein Z  Prolonged in deficiencies of fibrinogen,
prothrombin, V, VII, VIII, IX and X
Hemostasis Specimen Collection Tubes  Most sensitive to factors VII deficiencies
 Mostly used collection tube:  Most often used to monitor effects of therapy with
- Plastic blue stopper (blue-top, blue- the oral anticoagulant warfarin
closure) sterile evacuated blood
collection tube Warfarin therapy and the Prothrombin Time
Warfarin : Vitamin K Antagonist
 Tubes of uncoated soda-lime glass
 Warfarin sodium ( 4-hydroxycoumarin, Coumadin)
- Unsuitable; negative surface charge
 Oral anticoagulant therapy
activates platelets and plasma
- Prevents strokes
procoagulants - Prevents VTE after trauma and surgery
 Siliconized (plastic-coated) glass tubes - Inhibits recurrence of DVT or PE
- Available but not usually - Used as therapy after AMI if it is
recommended complicated by CHF or coronary
insufficiency
Hemostasis Specimen Collection Rules
Prothrombin components
 For multiple tube collection:
Prothrombin reagent components:
- Must be collected first after a
 Tissue factor
nonadditive tube (plain tube).
 Phospholipid
- Must not follow tubes that contain  Ionic calcium (calcium chloride)
heparin (green), EDTA (lavender), Prothrombin procedure
sodium fluoride (gray) or tubes with  Reagent warmed at 37C
clot activators (red or yellow)  Aliquot of plasma PPP (50 – 100 mcL) warmed at
 Ratio of blood to anticoagulant is 9:1 37C for within 3 – 10 mins.
 Using a winged – needle butterfly set:  Add 100 or 200 mcL to plasma PPP aliquot
- Collect and discard blood from a  Start timer
nonadditive tube or an identical blue  Stop timer as clot is formed.
topped tube.  Record time.
 Clotted specimens: not acceptable
Prothrombin Time QC
 Do not excessively agitate the blood or shake  Perform normal and prolonged controls for PPP at
the tube. the start of each 8hr shift or with every change of
 Avoid excessive manipulation of the needle reagent.
during blood extraction.  QC materials:
 Remove the tourniquet within 1 minute upon - Commercially prepared lyophilized control
application to avoid stasis PPPs
- Or, collect and pool PPP specimens from
designated subjects.
Hemostasis Specimen Management
QC to be considered valid:
 Storage:
 Normal QC: within the reference interval
- Sodium citrate – anticoagulated whole  Prolonged QC: within the therapeutic range for
blood specimens: warfarin
- stand in vertical position with stopper
intact. Reporting of Prothrombin Time Result
- Maintained at RT never at ref temp  Report result to the nearest tenth of a second along
and never at higher than 24C with the PT reference interval

CALIMBAS – MT1143
  If there are duplicate tests, average of the two test silica, kaolin, ellagic acid or celite in suspension
results should be reported. and calcium
 Activator = change in plasma Factor XII = XIIa
International Normalized Ratio (INR)  XIIa forms complex with HMWK and Pre-K
 Is being reported by laboratories for patients with  = in vitro clot formation through intrinsic pathway
stable anticoagulation response. but not part of in vivo coagulation
ISI
INR = (PT patient / PT geometric mean of normal)  Factor IXa + calcium + phospholipid + factor VIIIa =
 Where: complex = catalyzes factor Xa
- PT patient is the result of patient in seconds  Factor Xa = complex with calcium, phospholipid
- PT geometric mean of normal is the PT from and factor Va = catalyze conversion of factor II to
geometric mean of the reference interval IIa.
- ISI is the international sensitivity index  Factor IIa = fibrin polymerization
 End point is clot formation
Prothrombin Time Reference Interval
 Typically, reference interval is 12.6 to 14.6 seconds Prolonged PTT results
 It is recommended that each center must establish  Deficiencies with factors XI, IX, VIII, X, and V;
their own range for every new lot of reagent being prothrombin and fibrinogen
used.  Also prolonged in presence of LA
 Prolonged in the presence of anti – factor VIII
Use of Prothrombin Time antibody, antibodies to factor IX and other
 Used diagnostically when coagulopathy is coagulation factors.
suspected.  Prolonged in therapeutic heparin
 Sensitive to liver diseases  No effect in PTT: deficiencies in factor VII and XIII.
 Prolonged PT:
- Single factor deficiendies of factor X, VII or V Procedure
- Prothrombin deficiency  Warm 50 or 100 mcL reagent at 37C
- Fibrinogen deficiency  Warm equal volume of PPP
- Acquired multiple deficiencies such as DIC,  Incubate for 3 mins(usual)
liver dse’s and VitK def (affects factor VII  50 or 100 mcL of warmed 0.025 mol/L calcium
activity) chloride is added
 Start timer.
Vitamin K deficiency
 Stop the timer when clot is formed.
 Seen in severe malnutrition
 Record the time.
 During use of broad spectrum antibiotics that
 Manual procedures should be performed in
destroy the gut flora
duplicate
 During malabsorption syndromes. PTT: QC
PT is the best indicator
same with PT
Limitations of Prothrombin Time
PTT reference interval
Problem Solution
 Typical range: 26 – 38
Blood collection PT falsely prolonged; repeat
Establish own reference values
volume less than specimen collection
specified
Collection of blood for Coagulation Tests
Hematocrit more Adjust the anticoagulant volume
than 55% using formula:
-3 1. Two – syringe Method
C = (1.85 x 10 )(100-H)V
Then repeat specimen collection - Used in all coagulation tests except the
Clot in specimen All results are affected; repeat one-stage PT
collection - Obtain 2 – 3 ml of blood, then detach the
Visible hemolysis PT falsely shortened; repeat needle from the adapter / barrel then
collection attach a new barrel. Make sure the needle
Icterus or lipemia Measure PT using mechanical is not dislodged from the vein. ** better if
coagulometer you use evacuated tube and plain
Heparin therapy Use reagent known to be evacuated tube
insensitive to heparin or one that
includes a heparin neutralizer such
2. Silicon glasswares
as Polybrene
- Dry and sterile glasswares are immersed
Lupus anticoagulant PT result is invalid; use
(LA) chromogenic factor X assay instead in silicon concentrated solution for at least
of PT five minutes and then rinsed with DH2O
Incorrect calibration, Collect the analytical error then and air dried or dried in oven
incorrect dilution of repeat the test - Siliconizing the glasswares will make the
reagents surfaces smooth and this will prevent
hemolysis and coagulation of blood cells.
Partial Thromboplastin Time However, disposable test tubes, needles
and syringes and the use of contact
Principle activators in some of the coagulation tests
 PTT reagents: contains phospholipid (previously may not need siliconizing
called partial thromboplastin)
 Together with phospholipid, it also contains
negatively charged particulate activator such as
CALIMBAS – MT1143
 3. Test tubes Reptilase Time
- Should be made of plastic or of disposable  Reptilase – enzyme derived from snake
glass and should not be reused. venom:
- Tubes should be without scratches  Snake: Bothrops atrox
- If disposable glass tubes are to be used,
rinsed first with DH2O and air dry before Principle:
using.  Reptilase enzyme cleaves only fibrinopeptide
A from the fibrinogen molecule to form firbin
Fibrinogen level determination
Two methods used in fibrinogen assay: Results:
 Normal Value is 10 – 15 seconds
1. Turbidimetric method
- precipitates the fibrinogen from the Prolonged in:
plasma by ammonium sulfate and the  Fibrinogen Deficiency
turbidity is measured  Presence of Fibrin Degradation Product and
Fibrin Split Product
2. Fibrin Clot method  Presence of streptokinase
- More accurate, rapid and convenient.
- The fibrinogen in the plasma is converted **Unaffected by Heparin
into a fibrin clot by the addition of
 Increase TCT and normal RT indicates
thrombin. The clot is separated and
presence of heparin
washed with NSS to remove other
proteins, and the fibrinogen is determined
Stypven Time / Russell Viper Venom Time
as protein with a biuret reagent.
 NV: 6 – 10 secs
 From East India Viper
Normal value: 200 – 400 mg / 100 mL plasma
 Venom directly activates factor X
 Determination of coagulation factor deficiency
Thrombin Clotting Time Reptilase Time
involved in the common pathway (X-V-II-I)
Chap 45 (Lab) Problem:
 Increased PT and increased Stypven
Thrombin Clotting Time
- Common (X-V-II-I)
 Increased PT and normal Stypven
Reagent:
- Extrinsic (VII)
 Commercially prepared bovine thrombin
reagent at 2 NIH units/mL
Coagulation System
Procedure:
1. Pre-warm (37C) the reagent within 3 to 10 Customary Name of Procoagulants
 FACTOR I – fibrinogen
mins.
 FACTOR II – prothrombin
2. Incubate (37C) 100 mcL of PPP within 3 to 10
 FACTOR III – tissue factor
mins
 FACTOR IV – ionic calcium
3. Pipette 200 mcL of thrombin in PPP and start
 FACTOR V – labile factor
timer.  FACTOR VII – stable factor
4. Stop timer once a fibrin polymer (clot) is  FACTOR VIII – antihemophilic factor
detected  VWF – Factor VIII carrier; platelet adhesion
5. Repeat the procedure to get duplicate results.  FACTOR IX – christmas factor
 FACTOR X – stuart-power factor
Quality Control:  FACTOR XI – plasma thromboplastin antecedent
Normal Control – results should fall within the (PTA)
laboratory reference interval  FACTOR XII – Hageman factor
Abnormal Control – result should fall within the range  Prekallikrein – fletcher factor; pre-K
of TCT in moderate hypofibrinogenemia  High molecular weight kininogen; fitzgerald factor;
HMWK
Results:  FACTOR XIII – fibrin-stabilizing factor (FSF)
 Platelet factor 3 – Phospholipids;
 Reference Interval is 15 – 20 seconds
phosphatidylserine, PF3
 Establish own interval within laboratory
Classification of Procoagulants
Prolonged TCT: PHYSIOLOGIC FUNCTION OF PROCOAGULANTS
 When fibrinogen level is less than 100 mg/dL
(hypofibrinogenemia) Serine proteases or cofactors
 If there is presence of antithrombotic materials  Serine proteases:
◦ Thrombin (factor IIa), factors VIIa, Ixa, Xa,
 Afibrinogenemia
XIa, and XIIa; and prekallikrein (pre-K)
 Dysfibrinogenemia  Cofactors:
 Presence of heparin ◦ TF, Factor V, Factor VIII and HMWK

CALIMBAS – MT1143
Remaining components: INTRINSIC PATHWAY
 Fibrinogen  Reaction that begins with Factor XII and culminates
◦ ultimate substrate of coag pathway in fibrin polymerization
 Factor XIIIa  Coagulation factors: XII, pre-K, HMWK, XI, IX, VIII,
◦ transglutaminase X, V, prothrombin and fibrinogen
 Phospholipids & calcium
 VWF EXTRINSIC PATHWAY
◦ large glycoprotein that participates in platelet  Factors VII, X, V, prothrombin and fibrinogen
adhesion
COMMON PATHWAY
BIOCHEMICAL NATURE OF SEVERAL  2 pathways have in common Factor X, Factor V,
PROCOAGULANTS prothrombin and fibrinogen
Vitamin K-dependent prothrombin group
 Prothrombin group Cell-based (In Vivo) Coagulation
◦ prothrombin; factors VII, IX, and X 3 SEPARATE BUT OVERLAPPING PHASES
 Regulatory proteins
◦ protein C, protein S and protein Z  INITIATION:
◦ Continuous low level activation of the TF
VWF:Factor VIII complex pathway occurs on the TF bearing cells outside
◦ VWF : this has receptor sites for platelets the circulation even in the absence of injury
and collagen
◦ Factor VIII : Produced by hepatocytes and  AMPLIFICATION
other tissues ◦ When injury and bleeding occurs, platelets
and VIII:VWF complex spill into the
Fibrinogen structure and Fibrin formation extravascular space
 Fibrinogen – primary substrate for thrombin
- Thrombin cleaves fibrinopeptides A  PROPAGATION
and B ⇨ fibrin monomer ⇨ fibrin ◦ Reaction occurs on the surface of the activated
polymer platelets w/c has all the components of
coagulation needed
Coagulation Pathways
TISSUE FACTOR PATHWAY Coagulation Regulatory Mechanisms
 TF – membrane receptor for factor VIIa
 inflammatory conditions – leukocytes and other Principal regulators:
cells expresses TF and initiate coagulation  Tissue factor pathway inhibitor (TFPI)
 Antithrombin
3 multimolecular complex formation  Protein C pathway
 TF, factor VIIa, phospholipid, Ca
◦ on the TF bearing cells Deficiency – associated with increased incidence of venous
 Factor IXa, Factor VIIIa, phospholipid, calcium thromboembolic disease
◦ On platelets, a.k.a tenase
 Factor Xa, factor Va, phospholipid, calcium Tissue Factor Pathway Inhibitor
◦ a.k.a prothrombinase  Factor VIIa:TF activates Factor X and IX
 Factor Xa reacts w/ factor VIIa:TF and binds TFPI;
CONTACT FACTORS important coagulation regulatory protein
 Contact factor complex composed of: Factor XIIa,  Protein S
HMWK, and pre-K activates factor XI ◦ Cofactor of activated Protein C (APC)
 Factor XII is activated in vitro by contact w/ ◦ Cofactor of TFPI; stimulates Xa inhibition
negatively charged surfaces  Because of TFPI, TF:VIIa:Xa reaction is short-lived
 In vivo, foreign materials and it may cause  Amplification of coagulation occurs primarily
thrombosis through IXa

FACTOR XI ACTIVATION PATHWAY Protein C Regulatory System

 Factor XI – activated by thrombin as well as contact  Intact blood vessels – thrombin binds the
factors endothelial cell membrane
 Thrombin-thrombomodulin complex activates
THROMBIN zymogen protein C ⇨ serine protease (activated
 Primary function – cleave fibrinopeptides A and B protein)
from the α and β chains of the fibrinogen molecule  protein APC-protein S complex ⇨ hydrolyzes and
triggering fibrin polymerization inactivates factors Va and V IIIa ⇨ slowing or
 Also activates Factor XIII to further stabilize the blocking coagulation
fibrin clot
 Chief protease of the coagulation pathway because Serine Protease Inhibitors (Serpins)
it plays a role in:  Antithrombin
◦ Coagulation  Heparin cofactor II
◦ Platelet activation  Z-dependent protease inhibitor (ZPI)
◦ Coagulation control (protein C)  Protein C inhibitor
◦ Fibrinolysis  α1-Protease inhibitor (α1-antitrypsin)
 Α2-Macroglobulin

CALIMBAS – MT1143
Coagulation Pathways Coagulation Pathways

Coagulation Pathways Coagulation Pathways

Coagulation Pathways Coagulation Pathways

Coagulation Pathways Coagulation Pathways

Coagulation Pathways
Coagulation Pathways

CALIMBAS – MT1143