(M) Lesson 7: Coagulation Tests (Part 1) Flashcards
What are the four (4) tests for intrinsic and common pathway?
- Lee and White whole blood coagulation time
- Plasma Recalcification Time
- Activated Clotting Time/Recalcification Time
- Partial Thromboplastin Time (PTT)
- Historically used as a screening test of all stages in the intrinsic pathway
- Time consuming, poor reproducibility, sensitive only to extreme factor deficiency
- Procedure: Three (3) glass tubes with venous blood placed in a 37 deg C water bath
- Amount of time to form a clot is recorded
Lee and White Whole Blood Coagulation Time
What is the principle for Lee and White Whole Blood Coagulation Time?
Length of time required for a measured amount of venous blood to form a solid clot
What is the reference range for Lee and White method?
5 to 15 minutes
T or F: The timer for the Lee and White method starts when blood appears in the hub.
T
Who discovered the Lee and White method?
Roger Lee and Paul White
The surface referred to when we say that if blood is transferred to a tube with parafilm, it does not clot.
Non-wettable surface
Discovered the concept of non-wettable surface
Paul Morawitz
T or F: The angle of tilt affects the results of the Lee and White method.
T
Give two (2) drawbacks of the Lee and White method.
- Lack of precision
- Lack of standardization
- The measure of the ability of the blood to clot
- Assess the secondary hemostasis, particularly the intrinsic and common pathway
Clotting Time
Bleeding Time or Clotting Time
First to have results?
Bleeding Time
Bleeding Time or Clotting Time
Measures primary hemostasis
Bleeding Time
Bleeding Time or Clotting Time
Measures secondary hemostasis
Clotting Time
What are the three (3) methods of assessing clotting time?
- Slide or Drop Method
- Lee and White Method
- Capillary Method
Slide or Drop Method
- Sterilize the finger or the earlobe with cotton moistened with ____. Allow it to dry.
- Make a puncture to a depth of ____. Start the stopwatch as soon as ____. Wipe off the first drop of blood with dry cotton.
- Place a ____ on a clean dry glass slide.
- After ____, draw a fine wire or pin through the drop of blood and gently lift the wire or pin.
- Repeat at ____ until tiny strands of ____ clings to the wire or pin.
- Sterilize the finger or the earlobe with cotton moistened with 70% alcohol. Allow it to dry.
- Make a puncture to a depth of 3 mm. Start the stopwatch as soon as blood appears. Wipe off the first drop of blood with dry cotton.
- Place a large drop of blood on a clean dry glass slide.
- After 3 minutes, draw a fine wire or pin through the drop of blood and gently lift the wire or pin.
- Repeat at 30 second intervals until tiny strands of fibrin clings to the wire or pin.
What is the normal value of the slide or drop method?
2 to 4 minutes
Lee and White Method
- Label ____ glass test tubes with patient name and number them, 1, 2, and 3.
- Perform a ____ venipuncture using a ____ and drawn ____ of blood.
- Remove the needle from the syringe, and fill each of the three tubes with ____ blood.
- Place the three test tubes in a ____ water bath.
- At exactly ____, remove the first tube form water bath and tilt gently to a ____ angle to see whether the blood has clotted.
- If blood not clotted, return it to the water bath and examine it at ____ intervals.
- After the blood in the third tube has clotted, examine the ____ tube immediately.
- Then examine the ____ one.
- Record the time it took the blood in the ____ test tube to clot or average the clotting time of all tubes.
- Label three glass test tubes with patient name and number them, 1, 2, and 3.
- Perform a clean, untraumatic venipuncture using a 20-gauge needle and drawn 4 mL of blood.
- Remove the needle from the syringe, and fill each of the three tubes with 1 mL blood.
- Place the three test tubes in a 37°C water bath.
- At exactly 3 minutes, remove the first tube form water bath and tilt gently to a 45° angle to see whether the blood has clotted.
- If blood not clotted, return it to the water bath and examine it at 30 second intervals.
- After the blood in the third tube has clotted, examine the second tube immediately.
- Then examine the first one.
- Record the time it took the blood in the third test tube to clot or average the clotting time of all tubes.
What is the normal value for the Lee and White method?
7 to 15 minutes
This method utilizes a non-anticoagulated tube and is considered as dangerous.
Capillary Method
- Sterilize the finger or the earlobe with cotton moistened with cotton moistened with ____. Allow it to dry.
- Make a puncture to a depth of ____. Start the stopwatch as soon as ____. Wipe off the first drop of blood as soon as blood appears.
- Place the tube filled by capillary action for about ____ of its length.
- Lay the tube on the table.
- After ____, gently break off ____ cm of the filled end.
- Repeat at ____ intervals until coagulation has occurred. This is revealed by the presence of strands of ____, which span the gap at least ____ mm between the broken ends.
- Sterilize the finger or the earlobe with cotton moistened with cotton moistened with 70% alcohol. Allow it to dry.
- Make a puncture to a depth of 3 mm. Start the stopwatch as soon as blood appears. Wipe off the first drop of blood as soon as blood appears.
- Place the tube filled by capillary action for about 2/3 of its length.
- Lay the tube on the table.
- After 2 minutes, gently break off 1 cm of the filled end.
- Repeat at 30 second intervals until coagulation has occurred. This is revealed by the presence of strands of fibrin, which span the gap at least 5 mm between the broken ends.
What is the normal value of the capillary method?
2 to 4 minutes
Clinical Significance
Prolonged coagulation time is observed in what two (2) circumstances?
- Haemophilia
- Occasionally in diseases like obstructive jaundice, anemia, leukemia, hemorrhagic disease of the newborn and onset of severe acute fever
Match the following.
- Factor 8
- Factor 9
- Factor 11
A. Haemophilia B
B. Haemophilia C
C. Haemophilia A
- C
- A
- B
T or F: In the case of severe acute fever, the coagulation time of a patient does not go back to normal after the fever subsides.
F
- Modification of Lee and White
- Time required for specimen to clot after addition of calcium is recorded
Plasma Recalcification Time
Plasma Recalcification Time uses what type of plasma?
Citrated plasma
Plasma Recalcification Time
What is added to the citrated plasma for it to clot?
Calcium chloride
Match the following.
- PRP
- PPP
A. 130 to 240 seconds
B. 100 to 150 seconds
- B
- A
What type of plasma is used to know the platelet count?
PRP
What are the two (2) disadvantages of the plasma recalcification time?
- Difficulty in standardizing number of platelets in PRP
- Length of time in performing the test
- Modifies the plasma recalcification time
- Uses a special incubator (37 deg C) for blood to be kept warmed
- Principle: Whole blood contains all components to produce a clot when put into a glass tube. By adding an activator and keeping blood at 37 deg C, a more reliable screeen of intrinsic and common pathway is achieved.
Activated Clotting/Recalcification Time
Activated Clotting/Recalcification Time
- A diatomaceous earth used as a contact factor activator
- Utilized powdered form (can be performed bedside as POCT)
- More efficient in examining common and intrinsic pathway
Diatomite
- Measures factors in intrinsic and common pathway except factor VII and XIII.
Activated Partial Thromboplastin Time (APTT)
When there is a problem with both APTT and PT, it means there is a problem in ____ pathway.
Common
APTT is most useful in what two (2) circumstances?
- Screening for intrinsic pathway
- Monitoring heparin therapy (in thrombotic disorders to slow down clotting)
Specimen used for APTT?
Platelet Poor Plasma (PPP)
What are the three (3) reagents in APTT?
- Platelet substitute (phospholipid) from brain/plant phospholipid
- Activator (Kaolin, celite silica, ellagic acid)
- CaCl2
- Means that the reagent only contains the PL and no tissue factor
Partial thromboplastin
Principle of APTT?
Patient’s plasma + Ca + PL (partial thromboplastin) + activator (kaolin, celite silica, ellagic acid)
Normal value for APTT?
20 to 45 seconds
- Test of choice to screen for factor deficiencies of the intrinsic and common pathway
- Used to monitor heparin
Partial Thromboplastin Time (PTT)
APTT
What are the five (5) equipment and reagents?
- Phosphilipid with activators
- 0.025 CaCl2
- Controls
- Glass tubes
- Pipettes
APTT
- Collect blood specimen and place it on ____ top.
- Spin the blue top for ____ minutes at ____ spin.
- Separate the plasma to a clean tube and prewarm at ____ C.
- Simultaneously prewarm the ____ reagent and the ____ reagent at 37 deg C.
- Aspirate ____ or 100 uL of the prewarmed plasma and place it on another tube.
- Add 0.1 mL or ____ of the PTT reagent on the plasma and incubate the mixture for ____ minutes.
- Add 0.1 mL or 100 uL of ____ to the mixture of Plasma and PTT reagent, and start the time using the stopwatch.
- Check for the ____ by tilting the tube at an angle of approximately ____ degree.
- As the gel formation is observed stop the time and record the result.
- Collect blood specimen and place it on citrated top.
- Spin the blue top for 15 minutes at heavy spin.
- Separate the plasma to a clean tube and prewarm at 37 deg C.
- Simultaneously prewarm the reconstituted PTT reagent and the 0.025 M calcium chloride reagent at 37 deg C.
- Aspirate 0.1 mL or 100 uL of the prewarmed plasma and place it on another tube.
- Add 0.1 mL or 100 uL of the PTT reagent on the plasma and incubate the mixture for 3-5 minutes.
- Add 0.1 mL or 100 uL of 0.025 M CaCl2 to the mixture of Plasma and PTT reagent, and start the time using the stopwatch.
- Check for the gel formation by tilting the tube at an angle of approximately 40-45 degree.
- As the gel formation is observed stop the time and record the result
APTT is reported in ____, to the nearest tenth along with the reference range.
Seconds
Prolonged APTT in the absence of heparin use indicates?
A factor deficiency
Prolonged APTT may also be seen in patients with acquired circulating anticoagulants such as those with?
SLE
How do we differentiate factor deficiency and acquired circulating anticoagulant?
Mixing Studies/Substitution Studies
Series of mixing studies to find which clotting factor is deficient using correction reagents
Mixing Studies
What are the four (4) correction reagents for mixing studies?
- Normal fresh plasma
- Aged plasma
- Adsorbed plasma
- Aged serum
Match the following.
- Lacks no clotting factors
- Lacks II, VII, IX, X (Vitamin K dependent factors)
- Lacks I, V, VIII, X, XIII
- Lacks V and VIII
A. Normal fresh plasma
B. Aged plasma
C. Adsorbed plasma
D. Aged serum
- A
- C
- D
- B
Match the following.
- All except V and VIII
- Has VII, IX, XI, XII
- Has all clotting factors
- Has I, V, VIII, XI, XII
A. Normal fresh plasma
B. Aged plasma
C. Adsorbed plasma
D. Aged serum
- B
- D
- A
- C
What is used in adosrbed plasma hence why it is adsorbed?
Barium sulfate
Which two (2) correction reagents are more commonly used because of the number of factors they have?
Aged plasma and Adsorbed plasma
What are the three (3) sources of error for APTT?
- Sample collection and preparation
- Reagent preparation
- Instrumentation
What are the two (2) tests for the extrinsic and common pathway?
- Prothrombin Time
- Stypven Time
- Measures factors in the extrinsic and common pathway
- Choice of test for monitoring coumarin/warfarin therapy for vitamin K antagonists
Prothrombin Time (PT)
Match the following.
- Orally taken
- Through IV
A. Heparin
B. Coumarin
- B
- A
Which four (4) factors are depressed by vitamin K antagonists?
- II
- VII (first to be affected)
- IX
- X
Which is the only factor not measured by PT?
Factor IX
Prolonged or Shortened PT?
- Deficiencies in Factor I, II, V, VII, X
- Liver disease
- Vitamin K deficiency
- Circulating anticoagulant
Prolonged
Specimen used for PT?
Platelet Poor Plasma (PPP)
Reagent used for PT?
Thromboplastin
Tissue extract with tissue factor and phospholipid
Normal value for PT?
11 to 14 seconds
- Developed by WHO in order to standardize PT ratio
- Decreases/eliminates differences seen between labs
- Formula: (Patient’s PT/PT Control)^ISI
International Normalized Ratio
Why is there a need to standardize the PT ratio?
Due to different thromboplastin
What does ISI mean?
International Sensitivity Index
Index of the thromboplastin reagent
ISI
Healthy individuals should have an INR of what range?
0.8 to 1.1
If a patient is taking warfarin to prevent blood clots, doctors will most likely choose to keep their INR between what range?
2.0 to 3.0
Higher INR can result to (faster/slower) clotting of blood.
Slower
Prothrombin Time
- Collect blood specimen and place it on ____ top.
- Spin the blue top for ____ at ____ spin.
- Separate the plasma to a clean tube and prewarm at ____ C for ____
- Simultaneously prewarm the reconstituted PT reagent at ____ C for ____
- Aspirate 0.1 mL or 100 uL of the ____ and place it on another tube.
- Add 0.2 mL or 200 uL of the ____ on the plasma, and immediately start the time using the stopwatch.
- Check for the ____ by tilting the tube at an angle of approximately 40-45 degree.
- As the gel formation is observed, stop the time and record the result.
- Collect blood specimen and place it on citrated top.
- Spin the blue top for 15 minutes at heavy spin.
- Separate the plasma to a clean tube and prewarm at 37 deg C for 2-5 minutes.
- Simultaneously prewarm the reconstituted PT reagent at 37 deg C for 2-5 minutes.
- Aspirate 0.1 mL or 100 uL of the prewarmed plasma and place it on another tube.
- Add 0.2 mL or 200 uL of the PT reagent on the plasma, and immediately start the time using the stopwatch.
- Check for the gel formation by tilting the tube at an angle of approximately 40-45 degree.
- As the gel formation is observed stop the time and record the result.
What are the four (4) ways that PT is reported?
- Patient time (in seconds) with the reference range
- Patient time with the control time (in seconds)
- PT ratio
- Percent Activity
This method of reporting for PT has been proposed as the standard method of reporting.
INR
The results of PT may be interpreted as what two (2) results?
- Abnormality of one or more common or extrinsic coagulation factors
- Factor inhibitor
What are the three (3) sources of error for PT?
- Sample collection and preparation
- Reagent preparation
- Instrumentation
- Utilizes the powerful coagulant property of Russell’s Viper Venom
- No need to perform if PT is normal
Stypven Time
- Capable of bypassing activation of factor VII, directly activating factor X
- Acts as a thromboplastin-like substance that directly activated factor X
Russell’s Viper Venom
In Factor VII deficiency:
PT: (prolonged/shortened)
ST: Normal
Prolonged
What are the other five (5) tests involved in coagulation tests?
- Thrombin Time
- Reptilase Time
- Clot Solubility Test
- Factor Substitution Test/Mixing Studies
- Inhibitor Studies
- Measures conversion of fibrinogen to fibrin
- Principle: Addition of thrombin bypasses all coagulation reactions and converts fibrinogen to fibrin
- Increased in: Dysfibrinogenemia, hypofibrinogenemia, fibrin split products
Thrombin Time
Match the following.
- 15 minutes
- 5 minutes
- 30 minutes
A. Orange Top
B. Red Top
C. Gold Top
- C
- A
- B
- A thrombin-like enzyme isolated from the reptile Bothrops athrox
- Advantage: Not influences by heparin and immunologic AT-III
- Disadvantage: Greatly affected by dysfibrinogenemia
Reptilase Time
Normal value for reptilase time?
10 to 15 seconds
Study the table for the comparison of Thrombin Time and Reptilase Time.
Go nyo na mwa.
- Fibrin clot must be insoluble to urea or monochloroacetic acid
- Normal value: Clot should remain insoluble for 24 hours at 37 deg C
Factor XIII Assay (FSF)/Duckert’s Test/Clot Solubility Test
Initial PTT is corrected after adding activators Kaolin or Celite
Ellagic acid is not used
Fletcher Factor Assay