TEST FOR

SECONDARY
HEMOSTASIS
1. Clotting time
 Period time required for free form of blood to
clot after it has been removed from the body.
 Initiation of coagulation  visible clot
 Capillary blood mtd: 2-4 minutes
 Drop/slide
 Capillary tube / Dale & Laidlaw

Whole blood / Lee & White Mtd: 7-15 mins

Lee and white method
 Coagulation time of whole blood is the length of
time required for a measured amount of blood to
clot under certain conditions
 Uses Gray stoppered tube (diatomaceous earth)
 2. Prothrombin Time

 Uses
 Measures EXTRINSIC PATHWAY & COMMON PATHWAY.
 To monitor warfarin/Coumadin dosage, liver damage &
vitamin K status.
 NV: 10-12 seconds
 Rule: 30% of concentration of II,VII,X (Normal)
 Prolonged
 Fibrinogen deficiency (<80mg/dL)
 Dysproteinemias
 Vitamin K deficiency
 Disseminated intravascular coagulation
 Liver diseases
 (+) FSP (Fibrinogen split products)
International Normalized Ratio
 For patients with a stable anticoagulant response.
 For monitoring oral anticoagulant therapy
ISI: Calibration parameter that
defines the responsiveness of
the rgt relative to WHO
International Reference
Preparation
REFERENCE INTERVAL: PROTIME

 12.6 to 14.6 seconds

INR in absence of anticoagulant therapy: 0.8-1.2
INR in anticoagulant therapy: 2-3
Limitations
ANTICOAGULANTS
1. Warfarin
 - Vitamin K antagonist that interfere with normal synthesis of
factor II, VII, IX and X as well as CHON C & CHON S.
 - cause incomplete coagulation

 Onset of action: 8-12 hrs

 Maximum effect: 36 hrs
 Duration of action: 72 hrs
ANTICOAGULANTS
2. Heparin
 - the mainstay of immediate therapy for acute
pulmonary embolism.
 - may cause bleeding & thrombocytopenia
 3. PARTIAL THROMBOPLASTIN TIME
Aka: Activated partial thromboplastin time
Thismeasures the time required to generate thrombin & fibrin
polymers via INTRINSIC & COMMON pathways.

USES
 Determination of deficiency involving INTRINSIC / COMMON
pathways
 Monitor the effects of unfractionated heparin therapy.
(coronary artery bypass & cardiac kmparrilla
catherization)
PTT REAGENTS

 Phospholipid (partial thromboplastin)

 Activator (silica, kaolin, ellagic acid or
celite)
 CaCl2
REFERENCE INTERVAL

 26 to 35 seconds
 **therapeutic range: 60-100 seconds
4. THROMBIN TIME
 5 NIH unit/mL Bovine thrombin reagent
Reagent cleaves fibrinopeptides A &B

 thrombin reagent + PPP  (OD) Fibrin clot

 *reference interval: 15-20 seconds

 Length of time of fibrin clot to form is recorded
 sensitive in detecting heparin inhibition
 Prolonged:
 Fibrinogen level: 75-100 mg/dL
 Dysfibrinogenemia
 (+) Heparin
 (+) FDP
 (+) Streptokinase
5. Reptilase Time
 Reptilase- Venom of Bothrops atrox
 Reagent cleaves fibrinopeptide A ONLY

 diluted Reptilase + PPP  Fibrin Clot

 *insensitive to heparin but sensitive to dysfibrinogemia

 *detect hypofibrinogemia , dysfibrinogemia & presence of FDPs and
paraproteins
 NV: 10-15 secs
 Prolonged
 Decrease Factor I
 Dysfibrinogenemia

 Streptokinase

 (+) FDP : Fibrin degradation products

 6. Stypven Time/Russell Viper Venom TIme
 -East Indian Viper
 Determination of coagulation factor deficiency
involving COMMON PATHWAY

 *inc PT, inc stypven: COMMON

 *inc PT, Normal stypven: EXTRINSIC
 NV: 6-10 secs
 7. Fibrinogen Assay
 *clot based method of Clauss, modification of TCT

50 NIH unit/ml bovine thrombin reagent + 1:10 Owren

buffer PPP  Fibrin Clot

• Reference interval is 220 - 498 mg/dl

8. Duckert’s Test / 5M Urea Solubility Test

 Determination of Factor XIII deficiency

 Rgt: 5M urea
 Substitute for Urea: 1% monochloroacetic
acid
2% acetic acid

NORMAL: clot is insoluble to urea (24 hrs)

FACTOR XIII Deficiency: clot soluble in urea
PARTIAL THROMBOPLASTIN
TIME
PER GROUP: duplicate( same ptx) 1 Control

1 2 3

4 5 6

1 PTT rgt 1 PTT rgt 1 PTT rgt

1 CaCl2 1 CaCl2 1 CaCl2
 PROTIME N. Control Patient Spx
Ptx spx - 100 ul
N. Control 100 ul -
Incubate for 1 min
Pre warmed PT rgt
200 ul 200 ul
with Ca
Start the timer upon adding the PT rgt
STOP when fibrin clot was observe
 PARTIAL
N. Control Patient Spx
PROTHROMBIN TIME
Ptx spx - 100 ul
N. Control 100 ul -
Incubate for 1-2 mins
PTT rgt 100 ul 100 ul
Mix & incubate for 3 mins
Prewarm CaCl2 100 ul 100 ul
Start the timer upon the addition of CaCl2
Stop the timer upon the appearance of Fibrin Clot
Mixing studies
 Adverse thrombotic or obstetric events
 Prolonged PTT raises the presumption of an lupus
anticoagulant
 Means of detecting unfractionated heparin, most
often: TCT (Thrombin clotting time) or chromogenic
anti-Xa heparin assay
Mixing studies
 Includes a 37 C incubation step
 LA and specific inhibitors require an inhibition to
enhance their avidity.
 May use PTT reagent with intermediate LA sensitivity
but may also use PT reagent, DRVVT or an LA sensitive
low phospholipid PTT reagent
Note:
Tests Activation site
Kaolin clotting time and PTT (KCT/PTT) Factor XII
Dilute Russell viper venom test (DRVVT) Factor X
Dilute thromboplastin time (DTT) Factor VII
PTT mixing studies
 1:1 heparin free/neutralized patient plasma
with
 reagent poor normal plasma(PNP)

 PTT is corrected = coagulopathy

 PTT is not corrected = presence of inhibitors
PTT mixing studies: LA
 1:1 heparin free/neutralized patient plasma with
 reagent poor normal plasma(PNP)

 * if PNP correct the PTT + ptx is bleeding = coagulopathy is

presumed
 * if incubated PNP correct PTT + ptx is bleeding = inhibitor
deficiency is suspected
 * if not corrected + ptx is not bleeding = LA suspected