Coagulation Testing

Coagulation Testing
By:Diane Jette
BioMedica Diagnostics Inc.

Composition of Blood
* Formed Elements
o Erythrocytes (RBC)
o Leukocytes (WBC)
+ Neutrophils
+ Eosinophils
+ Basophils
+ Lymphocytes
+ Monocytes
o Thrombocytes (Platelets)
* Plasma
o 92% water
o 7 to 9 % of solutes are proteins
+ 55 to 60% Albumin, 15% Globulins, 4% Fibrinogen
o Non-protein nitrogen substance, Enzymes, Antibodies, Electrolytes, etc.
o Serum: No fibrinogen or Factors II, V and VIII

Hemostasis is the arrest of bleeding from an injured blood vessel
* Vasoconstriction and compression of injured vessels
* Platelets adhere to the site of injury and form a platelet plug
* Platelets release factors to augment vasoconstriction and initial vessel wall repair
* Platelets provide surface membrane sites and components for the formation of enzyme/cofactor complexes in blood coagulation reactions

Coagulation Reactions Lead to the Formation of a Blood Clot
* Two pathways: Intrinsic and Extrinsic - Coagulation Cascade
* Formation of a prothrombin activator - complex of Factor Xa, Factor Va and procoagulant phospholipid on surface of platelets.
* Prothrombin activator cleaves prothrombin into two fragments to give Thrombin.
* Thrombin cleaves small peptides from fibrinogen to form fibrin monomers that polymerize.
* Thrombin activates Factor XIII to cross-link the fibrin to form an insoluble clot.

Coagulation Cascade
* Intrinsic Pathway: (APTT)
o Factors VIII, IX, XI, and XII.
o Activated on surface of exposed endothelium.
o Complexes form on platelet phospholipids.
* Extrinsic Pathway: (PT)
o Factors IV, V, VII, X
o Activated by Tissue phospholipids (Tissue Factor or Tissue thromboplastin) released into blood as a result of tissue damage.
* Common Pathway (Thrombin Time)
o Factors I and II
o Leads to the formation of Fibrin Clot
o Thrombin time does not measure deficiencies in Intrinsic or Extrinsic pathway

The Role of Calcium
* Ca ions are needed for most of the reactions in the Coagulation Cascade
* Ca-chelating agents are used in vitro as anticoagulants (Citrate, EDTA, Oxalate)
* When Coagulation Factors are synthesized without Vitamin K they cannot bind Ca and lose enzymatic function

Regulatory Mechanisms
* Inhibition of Factor Activity
o Plasma protease inhibitors: anti-thrombin III (ATIII), *2-macroglobulin, *1 - antiprotease
o Heparin converts ATIII from a slow acting inhibitor to an instantaneous inhibitor of Thrombin, Factor Xa and Factor IXa
o Protein C and Protein S are serine proteases that cleave Factors VIII and Factor Va rendering them inactive

Fibrolysis
* Fibrin clot is degraded by protolytic enzymes and fragments dissolved in blood
* Process is catalyzed by Plasmin
* Plasminogen is converted to Plasmin
* Activation by tissue plasminogen activator (tPA) and urokinase
* Fibron degrades into large fragments X and Y then smaller fragments D and E

Regulation of Fibrolysis
* Plasminogen activator inhibitors (PAIs) and plasmin inhibitors slow the fibrolysis process
* tPA and urokinase have short half-lives and are rapidly cleared through the liver
* Unbound plasmin is instantaneously neutralized by 2-antiplasmin

Hereditary Coagulation Disorders
* Hemophilia A
o Factor VIII deficiency
o 80% of all Hemophilia cases
* Hemophilia B
o Factor IX deficiency
* Prolonged ATPP
o Recovered by dilution 1:1 with normal plasma
* Normal PT and Normal Bleeding Time
* Factor XI Deficiency
o 5 to 9% of European Jews
* 2-antiplasmin Deficiency

Acquired Coagulation Disorders
* Liver Disease
o Impaired clotting Factor synthesis
o Increased fibronolysis
o Thrombocytopenia
* Desseminated Intravascular Coagulation (DIC)
o Something enters the blood that activates factors
o Complication of obstetrics, infection, malignancy, shock, severe brain trauma
o Elevated PT, APTT, D-Dimer and other fibron degradation products

Circulating Anticoagulants
* Antibodies that neutralize clotting factor activity
* Factor VIII Anticoagulants
o Antibody
o Same profile as Hemophilia A
o Clotting time not restored by mixing with normal plasma
o Life-threatening condition

Lupus Anticoagulants
* Antibodies to phospholipid binding sites on clotting factors
* Prevent factors from accumulating on phospholipid surfaces
* Elevated APTT clotting times not corrected with mixing with normal plasma
* PT normal or slightly elevated.
* Non-specific depression of clotting factor activities (Factors VIII, IX, XI, XII)
* Test sensitivity increased by using diluted reagent
o Dilute ATPP reagent, Russell’s viper venom time, Kaolin time
o Clotting times corrected with the addition of phospholipids

Oral Anticoagulant Therapy
* Coumadin or Warfarin
* Inhibitor of Vitamin K dependant Factor synthesis
* Oral anticoagulant
* Dose regulated by therapeutic effect
* PT assay to measure INR
* INR range established for optimum therapeutic effect (typically 2.0 to 3.0)

Prothrombin Time: PT
* PT reagent contains Calcium ions and Thromboplastin from brain tissue (Rabbit).
* Thromboplastin (Tissue Factor) protein-lipid complex found in tissues outside blood vessels.
* Measures the function of the Extrinsic Pathway.
* Sensitive to Factors IV, V, VII, X.
* Provided as a lyophilized reagent.
* Used to monitor oral anticoagulant therapy (Warfarin / Coumadin).

PT Reagent Calibration
* Reagents are calibrated against standard PT reagent established by the WHO.
* ISI = International Sensitivity Index.
* ISI is assigned by the manufacturer for each lot of reagent using reference material traceable to WHO.
* The lower the ISI the more sensitive the Reagent
o ISI of 1.8 to 2.4 = Low sensitivity (North American Standard PT)
o ISI of 1.4 to 1.8 = Average sensitivity
o ISI 1.0 to 1.4 = High Sensitivity

PT: INR Values
* INR = International Normalised Ratio.
* MNP = Mean Normal Plasma.
* INR = (PT / MNP)ISI
* An INR of 1.0 means that the patient PT is normal.
* An INR greater then 1.0 means the clotting time is elevated.

INR Calculation
* Example 1
o MNP = 12.0 s
o ISI = 1.25
o Patient Plasma = 20 s
o INR = (20.0 / 12.0)1.25 = 1.9
* Example 2
o MNP = 12.0 s
o ISI = 1.85
o Patient Plasma = 17 s
o INR = (17.0 / 12.0)1.85 = 1.9
* Example 3
o MNP = 12.0 s
o ISI = 1.4
o Patient Plasma = 20 s
o INR = (20.0 / 12.0)1.4 = 2.0
* Example 4
o MNP = 12.0 s
o ISI = 2.0
o Patient Plasma = 20 s
o INR = (20.0 / 12.0)2.0 = 2.8

Expected PT Values
* Mean Normal Plasma = 10 to 14 seconds.
* Mean Normal Plasma value varies with PT sensitivity. A high sensitivity (Low ISI) PT will give a high normal PT value (13 to 15 seconds).
* Oral anticoagulant monitoring = Target INR of 2.0 to 3.0.
* INR of greater than 5 or 5.5 = unacceptable high risk of bleeding.
* %CV between duplicates less than 5%

Performing a PT test
* Pre-warm PT reagent and sample to 37 oC
* Add 100 L sample to cuvette
* Add 200 L of PT reagent to cuvette
* Start timer
* Record time to clot in seconds
* Calculate INR
* see product insert for PT

Activated Partial Thromboplastin Time
* APTT or PTT
* Reagent contains phospholipids and a ‘surface activator’; (Ellagic Acid, Micronized Silica)
* Calcium Chloride reagent added to start the reaction.
* APTT reagent mimics the surface of a platelet.
* Measures activity of clotting factors in the Intrinsic Pathway, factors VIII, IX, XI and XII
* No WHO calibration standard

Uses of APTT
* Sensitive to 30 to 40% deficiencies of all factors except VII and XIII.
* Heparin inhibits the activity of clotting factors in the Intrinsic Pathway.
* A standard curve (APTT time vs Heparin concentration) is prepared using a heparin standard.
* APTT is also sensitive to other non-specific Factor inhibitors such as Lupis Anticoagulant.
* Can be influenced by Vitamin K deficiency and Coumadin therapy.
* Negative APTT result usually rules out Hemophilia.

Expected APTT Values
* Normal Range: 26 to 40 seconds
* Slightly Elevated: 45 to 65 seconds
* Extremely Elevated = > 70 seconds
* %CV less than 7%

Performing an APTT Test
* Pre-warm Calcium Chloride reagent to 37 oC.
* Add 100 microL of sample to cuvette.
* Add 100 microL of APTT to cuvette and incubate for 3 minutes.
* Add 100 microL of Calcium Chloride reagent and start timer.
* Record the time to clot in seconds.
* See APTT product Insert

Heparin Monitoring
* Prepare Heparin Standards
o Prepare stock heparin (10 USP units/mL)
o Prepare working heparin dilutions (0.1 to 0.8 U/mL) by diluting stock in normal plasma
o Run heparin dilutions as samples in APTT assay
* Plot the results as U/mL vs Log Clotting times
* Run patient samples in APTT assay and determine heparin concentration from the plot.

Heparin Calibration Curve
Factor Substitution Tests:
PT and APTT
* Dilute patient sample one to one with adsorbed plasma and serum to determine if normal clotting time is restored.
* Serum: Source of Factors IX, X, XI & XII
* Adsorbed Plasma: Source of Factors VIII, V, XI, & XII

Factor Plots: PT and APTT
* Dilute a normal plasma sample or control 1 to 10 in saline
* Dilute in saline: 100%, 50%, 25%, 12.5%. 6.25%, 3.12%,
* Mix one to one with a factor deficient plasma.
* Measure PT or APTT and plot Log % Factor vs Log clotting time.
* Dilute patient plasma 1 to 10 in saline.
* Mix one to one with a factor deficient plasma.
* All factors will be restored except the deficient factor. If the factor is present in test sample no reduction in clotting will be seen. (Same clotting time as the 100% standard)
* Determine percent factor from standard curve.

Factor Calibration Curve
Plasma Controls
* Used to monitor assay performance (QC)
* Made from pooled normal human plasma with some factors selectively adsorbed.
* Three levels available:
o Level 1 control represents a normal plasma
o Level 2 represents a slightly elevated plasma (INR ~ 1.5)
o Level 3 represents a severely elevated plasma (INR ~ 2.5)
* Each laboratory should establish expected ranges for PT and APTT.

Thrombin Time (TT or TAT)
* Measures common pathway.
* Fibrinogen --> Fibron Clot
* Not sensitive to deficiencies in Intrinsic or Extrinsic pathways.
* Reagent consists of animal thrombin.
* Normal Clotting time is 15 seconds.
* If elevated sample is mixed one to one with normal and re-tested. If normal clotting is not restored then an anti-coagulant is present.
Bleed Time Test
* An incision is made
* Time to stop bleeding is measured
* Normal clotting time is 7.5 minutes

D-Dimer Test
* D-Dimer is a fibrin monomer
* Product of fibrolysis
* Latex agglutination assay is used
* Anti-D-Dimer antibody coated on micro-latex beads
* Cardiac Infarction Marker

Activated Clotting Time (ACT)
* Clotting time of whole blood in the presence of silica based activator.
* Normal clotting times = 90 to 170 sec.
* Used to monitor heparin doses from 1 to 10 U/mL (APTT is sensitive to heparin at 0.2 to 1 U/mL).
* Used with invasive procedures that require on-site adjustment of heparin and protamine dosage. (ex. Cardiopulmonary bypass surgery).
* Not amenable for use with an optical instrument, too cloudy.
* Also called HMT, Heparin Management Test

Fibrin-1 Clot Detection
LIGHT SOURCE
CUVETTE
DETECTOR
Clot Detection
Time in seconds
Optical Density
Change in slope > Threshold = CLOT

Coagulation Testing.ppt