EVALUATION OF

COAGULATION C

DISORDERS
By : B.R.Stephenson
Moderator : Dr.N.Bharat Rao, Professor of Pathology
Contents
• Haemostasis
• Coagulation Disorders
• Evaluation of Coagulation Disorders
• Pre-Analytic Variables
• Screening Tests
• Second Line Tests
• Further Workup
Haemostasis
• Def : Hemostasis is a precisely orchestrated process
involving platelets, clotting factors, and endothelium
that occurs at the site of vascular injury and culminates
in the formation of a blood clot, which serves to
prevent or limit the extent of bleeding
• General sequence of events leading to hemostasis:
• Arterioal Vasoconstriction
• Primary Hemostasis
• Secondary Hemostasis
• Clot stabilization and resorption
Secondary
Hemostasi
s
• Tissue factor is
exposed at the site of
injury
• Tissue factor binds
and activates factor
VII
• This sets into motion
a CASCADE of events
leading to thrombin
generation
• Thrombin cleaves
circulating fibrinogen
into insoluble fibrin
and also leads to
additional platelet
aggregation
THE COAGULATION CASCADE
Evaluation of
coagulation system
1. Screening Tests
a) Prothrombin Time (PT)
b) Activated partial thromboplastin time (aPTT)
c) Thrombin Time
d) Platelet Count
2. Second Line Tests
a) Correction tests using PT or aPTT
b) Reptilase or ancrod time
Pre-Analytic
Variables
• Venous Blood Collected should be collected
aseptically
• Anticoagulant of choice – 3.2% Sodium Citrate
(0.109M citrate solution)
• Volume of Anticoagulant
• For a 5ml blood sample can be adjusted according to
haematocrit value
• Volume of Blood
• One may choose to keep the anticoagulant volume
of 0.5ml constant and adjust the volume of added
blood according to the haematocrit
• Formula : Volume of blood = (60-haematocrit)/ 100 x
4.5
Pre-Analytic Variables
• Preparation of platelet-poor plasma
• Most routine coagulation investigations are performed on platelet-poor
plasma (PPP)
• Prepared by Centrifugation at 2000g for 15 min at 4oC, preferably in a
refrigerated centrifuge
• Samples should be kept at :
• 4oC – For most assays
• Room Temperature –Lupus Anticoagulant or Factor VII assays
• The testing should preferably be completed within 2 hours of collection
Pre-Analytic Variables
• Storage of Plasma (Deep freezing plasma)
• Optimum – in Liquid nitrogen (-196oC)
• Samples may be frozen at -40oC or -80oC for several weeks without significant
loss of most haemostatic activities.

• Sample Thawing
• Samples should be thawed at 37oC
• Gentle but thorough mixing of samples is essential after thawing and before
testing
• Once thawed, the sample should never be refrozen
Prothrombin Time
• Indicates the overall efficiency of the extrinsic clotting
system
• Sensitive to changes in factor V,VII and X
• Reagents
• Platelet poor plasma from the patient
• Control Plasma Sample
• Thromboplastin
• Calcium Chloride – 0.025 mol/lit
Prothrombin Time
• Results
• Results can be expressed as
a) Mean of duplicate recording in seconds
b) INR ( International Normalized Ratio) = (PT of patient / Control PT) ISI

• Normal Range
• 10-12 seconds (with recombinant human Thromboplastin)
• Each Laboratory should establish its own normal range
Interpretation
• Common Causes of prolonged PT
1. Administration of oral anticoagulant drugs (Vitamin K antagonists)
2. Presence of direct acting inhibitor of factor Xa
3. Liver disease
4. Vitamin K deficiency
5. Disseminated intravascular coagulation
6. Rarely, a previously undiagnosed factor VII, X, V or Prothrombin
deficiency
Activated Partial
Thromboplastin Time
• Measures the clotting time of plasma after the activation of
contact factors but without added tissue thromboplastin.
• Indicates the overall efficiency of the intrinsic pathway.
• It is also sensitive to circulating anticoagulants (inhibitors)
and heparin.
• Depends on contact factors, factors VIII, IX, X, V,
prothrombin and fibrinogen.
Activated Partial
Thromboplastin Time
1. Mix equal volumes of phospholipid reagent and the kaolin
suspension and leave in a glass tube in the water bath at
37 degree.
2. Place 0.1ml of plasma into a new glass tube.
3.Add 0.2ml of kaolin-phospholipid solution, mix the contents
and start the stopwatch simultaneously.
4. Leave at 37 degrees for 10mins with occasional shaking.
5.At exactly 10mins, add 0.1ml of pre-warmed CaCl2 and
start a second stopwatch.
6. Record the time taken for the mixture to clot.
7.Repeat the test on both the patient and the control plasma
atleast once.
Interpretation
• Normal range : 30 – 40 seconds
• A prolonged APTT with a normal PT indicated a possible deficiency of factor VIII, IX,
XI, XII, high molecular weight kininogen, prekallikrein or the presence of an
inhibitor.
• Causes of Prolonged APTT
• DIC
• Liver Diseases
• Administration/ Contamination of heparin
• A circulating anticoagulant
• Deficiency of coagulation factor other than factor VII
• Massive transfusion with stored blood
Thrombin Time
•Principle:
 The thrombin time reflects the reaction between thrombin and fibrinogen.
Thrombin
Fibrinogen Fibrin

 Thrombin is added to the plasma and the clotting time is measured.

 The Thrombin time is affected by the concentration and reaction of fibrinogen,
and by the presence of inhibitory substances, including fibrinogen/FDP and
heparin.
Thrombin Time
• Reagents
• Platelet poor plasma
• Thrombin solution which induces clotting of normal plasma in about 15 seconds

• Method
• Pipette 0.2ml test and control plasma into separate glass clotting tubes
• Warm to 37oC
• Add 0.1ml thrombin solution to each tube
• Start stopwatch for each tube
• Measure the clotting time and observe the nature of clot
• Repeat procedure for patient and control plasma
Interpretation
• Normal range : 15-19 seconds
• Causes of prolonged Thrombin Time
1. Hypofibrinogenaemia – DIC, congenital deficiency
2. Raised concentrations of Fibrin Degradation Product – DIC or Liver Disesase
3. Extreme prolongation of Thrombin Time – Unfranctionated heparin
4. Dysfibrinogenaemia – neonates , Liver disease
5. Hypoalbuminaemia
6. Paraproteinemia
Platelet Count
• Before considering further investigation of a suspected
bleeding disorder, always check the platelet count and the
blood film
• For Size and staining characteristics of Platelets
SECOND LINE
TESTS
C
Mixing
Tests
• Principle :
Prolongation of the
PT or APTT can be
investigated with
simple correction
tests by mixing the
patient’s plasma
with normal plasma.
• Correction 
possible factor
deficiency
• Failure to
correct 
presence of
inhibitor (inhibitor
screen should be
performed)
• Interpretation should
be cautious
Reptilase or Ancrod Time
• Reptilase – Bothrops atrox ; Ancrod – Agkistrodon rhodostoma
• Principle
• It is a serine protease, a thrombin like enzyme
• Cleaves fibrinopeptide A from fibrinogen (thrombin cleaves both A & B)
• Method
• Addition of reptilase to platelet poor plasma initiates clot formation
• Clot formation detected by optical/electromechanical methods

• The snake venoms are not inhibited by Heparin

Interpretation
• Reference interval : 18-22
seconds
• Causes of prolonged RT/AT
• Hypofibrinogenemia
• Dysfibrinogenemia
• Fibrin Degradation Products
• Other circulating anticoagulants
FURTHER WORKUP
C
• Investigation of a bleeding disorder resulting from a coagulation factor deficiency
• Automated Factor Assays
• One-stage assay of factor VII
• One-stage assay of factor VIII
• Two-stage and chromogenic assays for factor VIII
• Chromogenic factor IX assays
• Investigation of a patient with circulating anticoagulant
• Circulating inhibitor screen based on APTT
• Quantification of factor VIII inhibitors
• Fibrogen Estimation (Dry clot Weight)
• Defects of Primary Haemostasis
• Bleeding Time
• The PFA - system
• ELISA for von Willebrand factor antigen
• Ristocetin cofactor assay
• Investigations of a suspected disorder of Platelet function
References
• Robbins and Cotran, 9th Edition
• Dacie and Lewis, 12th Edition
• Williams Hematology, 9th Edition