Evaluation Tests for Secondary Hemostasis Principle:

 Measure the time required for clot to form after

Specimen Processing adding thromboplastin reagent with CaCl2 to
 4 hrs for APTT citrated platelet-poor-plasma
 24 hrs for PT  Run normal & abnormal controls
 Storage: PPP frozen at -20 degree C 1. 100 uL plasma
2. Incubate at 37®C for 2 mins
 Temperature at 37 degree C 3. 2OO uL Reaganet (0.025 M CaCl2 and Tissue
1. FV & FVIII breakdown above 37 degree C thromboplastin)
2. FVII & FXI activated at cold temperatures
 Reported in: clotting time in seconds, % activity &
Tests for Coagulation INR
1. Clotting time  Reference range: 10.0 to 14.0 seconds
2. Prothrombin Time  International normalized ratio
3. Activated Partial Thromboplastin time o Standardization of PT results reporting
4. Stypven Time/ Russell’s Viper Venom time o Independent of the reagent &
5. Thrombin Time instrument
6. Reptilase Time test o Set therapeutic range dependent on
7. Duckert’s test condition being treated: 2.0-3.0
8. Mixing study
𝑷𝒂𝒕𝒊𝒆𝒏𝒕 𝑷𝑻 (𝒔𝒆𝒄)
9. Fibrinogen Study INR= ( ) .𝑰𝑺𝑰
𝑴𝑵 𝑷𝑻 (𝒔𝒆𝒄𝒄)
10. Fibrinogen Level
11. Factor assays
 International normalized ratio
12. 5.0 M urea clot solubility test
Recommended Therapeutic Range
Condition: INR Ptx/Control PT ratio
Test for Fibrinolytic State
a) Prophylaxis of venous 2.0-3.0 1.2-1.5
1. Fibrin degradation products thrombosis in high risk
2. D-dimer assay individual or surgical ptx
b) Treatment of venous thrombosis 2.0-3.0 1.2-1.5
Clotting Time c) Prevention of embolism 2.0-3.0 1.2-1.5
 The time it takes for the clot to form d) Prevention of recurrent or ptx 3.0-4.5 1.5-2.0
 Blood is kept at 37 degree C with mechanical prosthetic
 Whole blood is placed in glass tube containing intravascular valve
activator

Drop or Slide Method

 Start from the last drop
 2-4 minutes

Clotting time
Capillary method
 2-4 minutes
Lee-White Whole blood clotting time
 5-15 minutes

Prothrombin Time
 Screening for extrinsic & common pathway
 Monitors anticoagulant therapy by Vitamin K
antagonists: Clinical Significance
o Warfarin, coumarin Prolonged PT:
 Reagents: 1. Factor deficiencies in extrinsic & common
1. Thromboplastin pathway
2. CaCl2 2. Factor activity less than 5-30%
3. Warfarin Therapy
 Sources of error Thrombin Time
 Falsely prolonged PT:  Tests the conversion of fibrinogen to fibrin
1. Unifilled tube  Qualitative/quantitative test for fibrinogen
2. Large clot in tube
3. Heparin contamination 1. 100 uL citrated platelet poor plasma
4. Hematocrit >55% 2. 200 uL thrombin CaCl2
5. Lipemia/icterus  Reference range: 10-14 seconds
 Relationship with fibrinogen level?
 Falsely short PT:
1. Small clot in tube Clinical Significance
 Prolonged TT:
Activated Partial Thromboplastin Time 1. Heparin, streptokinase therapy
 Screening for intrinsic & common pathway 2. Pregnancy, newborns
 Monitors unfractioned heparin therapy: 3. Degradation products, fibrin split products
4. Hypofibrinogenemia
 Reagents: 5. Afibrinogenemia
1. Plt phospholipid substitute 6. Dysfibrinogenemia
o Kaolin, celite, selica, ellagic acid 7. Multiple myeloma
2. CaCl2 8. Thrombin Inhibitor

 Run normal & abnormal controls Reptilase Time

 Not affected by the therapeutic heparin
1. 100 uL Plasma o Reptilase
2. 100 uL reagent (Phospholipid & activator-  Thrombin-like enzyme from
kaolin,celite,ellagic acid,silica) Bothrops atrox snake
3. 100 uL 0.025 m CaCl2 o Platelet-poor plasma + reptilase
 Reference range: 18-20 seconds
 Reference range: 23-35 seconds  Prolonged RT: hypofibrinogenemia,
afibrinogenemia,dysfibrinogenemia, FSPSs,
Clinical Significance streptokinase activity
 Prolonged aPTT:
1. Factor deficiencies in intrinsic & common Thrombin Time vs Reptilase Time
pathway Thrombin Time Reptilase Time
2. Factor activity <25-30% FSPs Prolonged Prolonged
3. Acquired circulating inhibitor Hypofibrinogenemia Prolonged Prolonged
o Heparin Dysfibrinogenemia Prolonged Prolonged
o Lupus inhibitor Immunologic anti- Prolonged Normal
o Antibody specific to a factor thrombins
Heparin Therapy Prolonged Normal
Sources of error
 Falsely prolonged aPTT: same with PT Stypven Time
 Falsely short aPTT:  Russell’s Viper Venom Time
1. Hemolysis  Derived from East Indian Viper, Vipera
2. Small clot in tube russelli
3. Plasma containing platelets  Based on activation of Factor X
 Reagent Preperation o Prolonged: neutralization of venom by
1. Incorrect dilution lupus inhibitor
2. Water impurities
3. Improper storage 1. 100 uL citrated platelet-poor plasma
 Instrumentation 2. Stypven reagent
1. Temperature  Reference range: 6-10 seconds
2. Light source
3. Bubbles in sample
Duckert’s Test Clot-based Assay Diagnosis
 5.0 M urea clot solubility test  Prothrombin time
 Tests for Factor XIII o Factor VII
1. Plasma is clotted  Vit K deficiency; malnutrition,
o PPP + 0.025 M CaCl2 malabsorption, newborns, hemorrhage
o Incubate for 1 hr at 37®C  Liver dse
2. Immersed in 5 M urea 1% monochloroacetic acid  DIC
 Normal: the clot should remain insoluble for 24 o Thromboplastin should be insensitive to
hrs at 37® C heparin
 Polybrene
Mixing & Correction Studies o Sensitive to lupus anticoagulant: anti-
 Performed when both PT & aPTT are prolonged to phospholipid antibody family
differentiate a factor deficiency from a circulating  Chromogenic factor X assay
inhibitor  Prolonged Aptt
o Factor VIII, IX & XI less than 0.3 U/ml
PT Abn Normal plasma Corrected o Factor XII, PK, HMWK
APTT Abn Adsorbed Plasma Corrected  Less than 30%
TT Abn Aged Serum Not corrected o Fibrinogen less than 100 mg/dl
o Lupus anticoagulant, anti-factor VII & IX
 Indicators o DIC, Vitamin K deficiency
o Corrections (shortened than reference  Thrombin Clotting Time
range) o Fibrinogen levels less than 100 mg/dl
 Factor deficiency o FDPs, paraproteins, heparin (antithrombotic
1. Hereditary activity must be ruled out)
2. Acquired o POCT
 Warfarin Therapy
 Liver Dse Fibrinogen Assay
o Partial or No correction  25 x more concentrated thrombin reagent
 Circulating inhibitor: heparin,  PPP diluted with Owren’s buffer
lupus inhibitor, VIII & IX o Minimizes antithrombotic effect of heparin.
inhibitor FDPs, paraprotein
Differential Diagnosis of Abnormal Coagulation Screening Tests o 1:10 Dilution of PPP to Owren’s buffer
Abnormal Activated Partial Thromboplastin Time (Aptt) alone o Not affected:
Associated with bleeding; VIII, IX and XI detectys 1. Heparin less than 0.6 U/ml
Not associated with bleeding; XII prekalikren (PK), HMWK, lupus 2. FDP levels less than 100 ug/dl
anticoagulants 3. Fibrinogen 150 mg/d’ or greater
Abnormal Prothormbin Time (PT) alone  Significance:
Factor VII defects
o Afibrinogenemia
Combined Abnormal APTT and PT
Medical conditions; Anticoagulants, disseminated intravascular
 Hemorrhagic disorder
coagulation (DIC), liver dse, Vit K deficiency, massive transfusion o Dysfibrinogenemia
Rarely dysfibrinogenemia; Factor X, V and II defects  Thrombosis

Specimen Management Factor Assay

 Specimen storage time & temperatures Factor System
 PRP= plt count 200-300 x 109/L Fibrinogen TCT
Prothrombin PT
 PPP= less than 10 x 109 /L
V PT
o Repeated thawing:
VII PT
 20®C for 2 weeks & less than -70®C for 6
VIII PTT
months or less IX PTT
 Thawed rapidly at 37®C X PT
 Mixed well & tested within 1 hr after removal XI PTT
from freezer XIII
 If not, store at 2-4®C for 2 hrs after thawing
 Bethesda Titer Plasminogen Assay
 Confirm & quantify specific anti-factor VIII  Chromogenic substrate assay
inhibitor 1. Amidolytic substrate as polypeptide Val- Leu-
 FVIII inhibitor neutralizes normal PPP=% activity Lys
of inhibitor 2. Add fluorophore or chromophore (pNA) to Val-
1. 0.2 ml of PPP with FVIII inhibitor + 0.2 ml Leu-Lys pNA in the carboxyl end
reagent with normal PPP 3. Hydrolysis of plasmin activated by
2. Incubated for 2 hrs at 37®C streptokinase
o 75% residual FVIII of the control: no  Yellow product
significance FVIII inhibitor
o 25%: FVIII inhibitor levels is tittered  Chromogenic substrate assay
o Reference range= 13.5 mg/dl
D-dimer Immunoassay
 Turbidimetry & nephelometry, etc. ↓Decreased in:
 Monoclonal anti-D dimer Ab 1. Thrombolytic therapy
agglutination reaction with ptx plasma D- 2. DIC
dimer 3. Hepatitis
4. Cancer
 Significance 5. Thrombotic disorders
o Venous thromboembolic dse ↑Increased in:
o Detection & monitoring of DIC 1. Inflammation
o Detection of myocardial infarction & 2. Pregnancy
ischemic stroke 3. Hemorrhage
o Thrombolytic therapy 4. Systemic fibrinolysis

 Turbidimetry & nephelometry tPA & PAI-1 Assay

 Cut-off varies on methodology  Chromogenic substrate assay
1. 0-250 ng/ml o tPA
2. 0-500 ng/ml 1. Reagent plasminogen + plasma TPA
3. 10-200,00 ng/ml 2. Plasmin is measured by chromogenic
assay
FDPs Assay
 Dtection FDPs at 2 mg/dl or greater  1.1 IU/ml upper limit
o Agglutiantions techniques  MI, stroke, DVT
o Polystyrene latex coated with anti-E and
Anti-D specific polyclonal Ab o PAI-1
1. tPA: PAI-1 conplex + residueal tPA
 3 samples: 2. Residual tPA activates plasminogen
o Undiluted to plasmin
o 1:5 3. Plasmin is measured by chromogenic
o 1:20 assay
 Results  10 ug/L
Result Agglutination  MI. DVT
2 ug/ml – 10 ug/ml Undiluted sample Euglobin clot lysis time
10 ug/ml- 80 ug/ml Undiluted and 1:5 o Inhibitor of fibrinolysis are removed
80 ug/ml above Undiluted,1:5 & 1:20 o Reaction of fibrinogen, plasminogen &
plasminogen activators & tPA
 Positive tests:  Steps:
1. Deep vein thrombosis 1. Preparation of euglobin fration
2. Pulmonary embolism  Diluted plasma acidified to pH 5.35 to
3. Systemic fibrinolysis 5.40 by 1% acetic acid
 Refrigeration for 30 minutes to
precipitate the reactions
 Centrifugation to obtain precipitate
 2. Precipitate dissolved in borate buffer
3. Add reagent thrombin or CaCl2
4. Observe clot for dissolution over 2 to 10 hrs

 Results
 < 2 hrs dissolution: excess fibrinolysis
 >10 hrs dissolution: deficient fibrinolysis

 Correlations
1. Depleted plasminogen: effect on dissolution?
2. Hypofibrinogenemia:
3. Deficient factor XIII

 Falsely prolonged:
1. Inflammation
2. Fibrinogen > 600 mg/dl
 WBCLT
o Stable 48 hrs
o <48 increased systemic fibrinolysis
 Protime sulfate (Plasma + protime sulfate)
 Ethanol gelation (Plasma +NaOH+ETOH)
o Fibrin monomers
o Fibrin strands (paracoagulation, gel-like
molecule)