Coagulation Testing Study Notes
COAGULATION TESTING
SPECIMENS FOR COAGULATION TESTING
Purpose: Assesses in vitro coagulation.
Lab Testing: Measures the time required to form a clot.
Pre-analytical Variation: Coagulation assays are highly sensitive to variations before testing; thus, collection must be meticulously performed.
Collection Protocol:
Avoid: Tissue thromboplastin contamination, inappropriate anticoagulant contact, usage of incorrect anticoagulant, improper temperature conditions, and hemolysis to prevent adverse outcomes for the patient.
ANTICOAGULANTS
Blood Type: Coagulation testing is conducted on plasma.
Anticoagulant: 3.2% sodium citrate is used.
Mechanism: Citrate binds to calcium ions, inhibiting multiple steps in hemostasis, thereby preventing the blood from clotting.
Collection Guidelines:
Avoid Glass Tubes: They may activate factors XI & XII during blood draw.
Blood to Anticoagulant Ratio: 9 parts blood to 1 part sodium citrate.
Vacuum Collection: Tubes must be collected via vacuum to the designated fill line.
Using Expired Vacutainers: This is prohibited.
Clotted Specimens: Such specimens are unacceptable for assays.
COLLECTION & STORAGE
Importance of Preventing Carryover: Essential to avoid contamination from tubes containing other anticoagulants.
Collection Order: Coagulation tubes should be collected first or only after tubes without additives.
Venipuncture Technique: Clean and rapid to avoid contamination with tissue thromboplastin.
Avoid Hemolysis: This can activate coagulation factors.
Temperature Regulations:
PT specimens may be stored at room temperature for up to 24 hours.
APTT specimens can only remain at room temp for up to 4 hours.
Butterfly System Usage: If this system is utilized, a discard tube must be used to prevent air from entering the vacutainer.
SPECIMEN PROCESSING
Transportation: To the lab must be quick and careful.
Centrifugation:
Samples should be centrifuged to obtain platelet-poor plasma (with PLT count < 10 x 10^9/L).
APTT specimens need centrifugation within 1 hour post-collection; PT specimens can stay uncentrifuged up to 24 hours.
Testing Protocol: If testing is done immediately, plasma can be retained on packed red cells. Otherwise, plasma must be separated from cellular components.
Tubes: They should stay stoppered until testing; unsuitable specimens for testing include lipemic, icteric, hemolyzed, or clotted specimens.
ERRORS AFFECTING TESTING
Common Errors:
Blood drawn into an incorrect tube type.
Incorrect plasma to citrate ratio caused by underfilling or a hematocrit exceeding 0.550.
Heparin contamination from IV contamination or incorrect order of draw.
Clotting in the tube attributable to traumatic venipuncture or improper mixing.
PERFORMANCE OF COAGULATION ASSAYS
Laboratory Protocol:
Adhere closely to each instrument's manufacturer instructions.
Normal and abnormal quality controls must be executed when testing commences each day, at the beginning of new shifts, or with every test run.
Controls are reconstituted daily from lyophilized or frozen specimens; once prepared, controls cannot be refrozen.
Quality controls (QC) should always be conducted under the same conditions as patient samples.
Laboratory Variability: Reference ranges are specific to each lab and should be reassessed with new reagent lots, instruments, or changes in collection methods.
CLOT ASSAYS
Factors Addressed: Prothrombin time (PT) and activated partial thromboplastin time (APTT) form the basis for screening tests for the coagulation system's functionality.
Follow-up Testing: If screening tests yield abnormal results, specific factor assays are performed.
PROTHROMBIN TIME (PT)
Overview:
Primarily screens the function of extrinsic and common pathways of coagulation, addressing factors VII, X, V, thrombin, and fibrinogen.
Commonly used for pre-operative screenings and monitoring of Warfarin (Coumadin) and other vitamin K antagnostic therapies.
Testing Method: Calcium and an excess of thromboplastin are added to patient plasma; the timing for clot formation is measured.
PRINCIPLE OF PT TEST
Reagents Required:
Prothrombin reagent, a commercial preparation of tissue thromboplastin.
Process: Combine prothrombin reagent with patient plasma.
Activation: Triggers the extrinsic pathway where prothrombin converts to thrombin and fibrinogen to fibrin clot.
The timing from thromboplastin addition to the formation of the fibrin clot is recorded.
Detection: Clot formation can be inferred from increasing sample viscosity and turbidity, with endpoint determination via mechanical or optical methods.
EXTRINSIC PATHWAY
Involved Factors:
III (Tissue thromboplastin)
VII activated:
Ca++
Xa
X
Ca++, PF3
V
Va
Prothrombin (II)
Fibrinogen (I)
XIII
Thrombin
XIIIa
Fibrin Formation:
Monomer forms stable fibrin (polymer).
REPORTING & REFERENCE INTERVALS
Normal PT Range: 10 – 13 seconds.
Therapeutic Range: Greater than 25 seconds.
Reported as the International Normalized Ratio (INR).
Factors Influencing INR:
Patient age, body mass, liver function, nutritional status, and genetics.
INR Calculation: Standardizes PT reporting across different labs with varying patient demographics; each reagent batch has an assigned International Sensitivity Index (ISI).
INR Reference Range: 0.9 – 1.13.
Increasing INR correlates to increased anticoagulation and is elevated in the presence of extrinsic factor deficiencies due to liver disease or when using oral anticoagulants.
INR Calculation Example:
Given: Patient prothrombin time is 23 seconds, mean normal protime of facility 10.5 seconds, ISI = 1.2.
Calculation:
Rounded: .
CONDITIONS AFFECTING PT
Normal Results:
Factor VIII: Normal
Factor XI: Normal
Factor XII: Normal
Prolonged Results:
Thrombin (II): Prolonged
Factors V, VII, X: Prolonged
Warfarin and Heparin therapy: Prolonged
Liver disease: Prolonged
Vitamin K deficiency: Prolonged
Underfilled tube: Prolonged
ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)
Screening Function: Evaluates factor deficiencies within intrinsic and common pathways, primarily for factors VIII, IX, XI, and XII.
Clinical Use: Commonly employed in pre-operative screenings and for monitoring heparin therapy.
TEST THEORY FOR APTT
Reagent Composition: Calcium chloride and partial thromboplastin (phospholipid component).
Process:
Add partial thromboplastin reagent to warmed patient plasma and activate the intrinsic pathway by adding CaCl2 to form the fibrin clot.
Calcium in blood binds to sodium citrate during specimen collection, losing calcium upon centrifugation, necessitating addition of these reagents for clotting.
INTRINSIC PATHWAY
Mechanism Overview:
Surface contact activates Factor XII with subsequent cascade involvement of Factors XI, IX, and VIII leading to thrombin production via the common pathway.
REPORTING & REFERENCE INTERVALS FOR APTT
Normal APTT Range: 25 – 40 seconds, with <35 seconds being desirable for surgical candidates.
Quality Control (QC):
Must be executed every 8 hours, abnormalities may surface from reagent deterioration, temperature variances, or instrument issues.
CONDITIONS AFFECTING APTT
Normal Results: Factors VII, XIII.
Prolonged Results: Factors I, II, V, VIII, IX, X, XI, XII with instances of both heparin and liver disease.
Underfilled Tube: Can also prolong APTT.
MIXING STUDIES
Purpose: Used for patients showing abnormally high PT or APTT.
Process:
An equal volume of patient plasma is combined with normal pooled plasma (1:1 mix) and re-tested for PT or APTT.
Interpretation:
If result normalizes, indicates factor deficiency or dysfunction. If unchanged, the specimen may contain inhibitors (e.g., heparin, oral anticoagulants, autoantibodies).
D-DIMER ASSAY
Definition: A degradation product of fibrin released during fibrinolysis by plasmin.
Usage: Primarily to rule out disseminated intravascular coagulation (DIC).
Reference Range: <500 ng/mL.
DIMER TESTING TECHNIQUES
Immunoturbidimetry (ELISA): Highly sensitive; capable of ruling out deep vein thrombosis and pulmonary embolism.
Particle Agglutination: Used for DIC detection; however, less sensitive, detecting dimers only at levels of 500 ng/mL.
ELEVATED DIMERS
Potential causes include:
Surgery, Trauma, Infection, Pregnancy, Cancer, Liver disease, Increasing age, Hospitalization.
The test shows sensitivity but lacks specificity.
THROMBIN TIME
Function: Assesses fibrinogen deficiencies or dysfunction and detects thrombin inhibitors, often elevated with heparin therapy.
Process: Patient plasma is mixed with thrombin and the time taken for clot formation is recorded.
FIBRINOGEN
Assessment: Activity levels of fibrinogen, with low levels indicating excessive loss (bleeding), consumption (DIC), or decreased production (severe liver disease).
Measurement Technique: Clauss method involves adding plasma to high thrombin concentrations, measuring clotting time, which is inversely proportional to fibrinogen activity (longer clot time = lower fibrinogen level).
ACTIVATED CLOTTING TIME (ACT)
Purpose: Non-automated test utilizing whole blood mixed with a clot activator.
Usage: Common in point-of-care testing and to monitor high-dose heparin therapy during surgical procedures.
Reference Range: 70 – 180 seconds.
COAGULATION TESTING METHODS
Methods Used:
Visual, Mechanical, Magnetic, Optical.
TESTING METHODS
Manual/Visual Method: Adding plasma and reagents to a glass tube and tilting to detect clot formation.
Mechanical Method: Employing warmed reagents, where a moving magnetic ball indicates clot formation.
Optical Method: Using photometric methods which isolate movement detection during clot formation.
AUTOMATION IN TESTING
Advantages: Capable of conducting large volume tests simultaneously with various assays.
Technology Used: Employs photometry or nephelometry to measure light scatter during clot formation.
Endpoints: Automated systems read optical density changes that indicate clot integrity, ensuring reagents and plasma are maintained at optimal temperatures.
CHROMOGENIC METHODS
Use: Synthetic chromogenic substrates are utilized to assess the activity of coagulation inhibitors on Factor X.