Coagulation Testing

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Coagulation Testing

• Diane Jette
• BioMedica Diagnostics Inc.

2
Composition of Blood

• Plasma
• 92 water
• 7 to 9 of solutes are proteins
• 55 to 60 Albumin, 15 Globulins, 4 Fibrinogen
• Non-protein nitrogen substance, Enzymes,
  Antibodies, Electrolytes, etc.
• Serum No fibrinogen or Factors II, V and VIII

• Formed Elements
• Erythrocytes (RBC)
• Leukocytes (WBC)
• Neutrophils
• Eosinophils
• Basophils
• Lymphocytes
• Monocytes
• Thrombocytes (Platelets)

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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

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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.

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Coagulation Cascade

• Intrinsic Pathway (APTT)
• Factors VIII, IX, XI, and XII.
• Activated on surface of exposed endothelium.
• Complexes form on platelet phospholipids.
• Extrinsic Pathway (PT)
• Factors IV, V, VII, X
• Activated by Tissue phospholipids (Tissue Factor
  or Tissue thromboplastin) released into blood as
  a result of tissue damage.

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Coagulation Cascade

• Common Pathway (Thrombin Time)
• Factors I and II
• Leads to the formation of Fibrin Clot
• Thrombin time does not measure deficiencies in
  Intrinsic or Extrinsic pathway

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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

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Regulatory Mechanisms

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

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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

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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

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Hereditary Coagulation Disorders

• Hemophilia A
• Factor VIII deficiency
• 80 of all Hemophilia cases
• Hemophilia B
• Factor IX deficiency
• Prolonged ATPP
• Recovered by dilution 11 with normal plasma
• Normal PT and Normal Bleeding Time
• Factor XI Deficiency
• 5 to 9 of European Jews
• ?2-antiplasmin Deficiency

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Acquired Coagulation Disorders

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

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Circulating Anticoagulants

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

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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
• Dilute ATPP reagent, Russells viper venom time,
  Kaolin time
• Clotting times corrected with the addition of
  phospholipids

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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)

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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).

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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
• ISI of 1.8 to 2.4 Low sensitivity (North
  American Standard PT)
• ISI of 1.4 to 1.8 Average sensitivity
• ISI 1.0 to 1.4 High Sensitivity

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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.

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INR Calculation

• Example 1
• MNP 12.0 s
• ISI 1.25
• Patient Plasma 20 s
• INR (20.0 / 12.0)1.25 1.9
• Example 2
• MNP 12.0 s
• ISI 1.85
• Patient Plasma 17 s
• INR (17.0 / 12.0)1.85 1.9

• Example 3
• MNP 12.0 s
• ISI 1.4
• Patient Plasma 20 s
• INR (20.0 / 12.0)1.4 2.0
• Example 4
• MNP 12.0 s
• ISI 2.0
• Patient Plasma 20 s
• INR (20.0 / 12.0)2.0 2.8

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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

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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

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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

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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.

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Expected APTT Values

• Normal Range 26 to 40 seconds
• Slightly Elevated 45 to 65 seconds
• Extremely Elevated gt 70 seconds
• CV less than 7

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Performing an APTT Test

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

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Heparin Monitoring

• Prepare Heparin Standards
• Prepare stock heparin (10 USP units/mL)
• Prepare working heparin dilutions (0.1 to 0.8
  U/mL) by diluting stock in normal plasma
• 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.

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Heparin Calibration Curve
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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

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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.

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Factor Calibration Curve
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Plasma Controls

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

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Thrombin Time (TT or TAT)

• Measures common pathway.
• Fibrinogen --gt 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.

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Bleed Time Test

• An incision is made
• Time to stop bleeding is measured
• Normal clotting time is 7.5 minutes

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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

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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

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