(M) Lesson 7: Coagulation Tests (Part 2) Flashcards
T or F: One of the most important tests in hemostasis is to assess the ability of clotting factors to do their functions.
T
- Also known as substitution test, mixing text, or factor identification
- Could identify which factor is missing by mixing correction reagents with plasma to determine if there is really an abnormality and then proceeding with PT and PTT
- Normal Values: Expected to lie around the reference range of PT and PTT
- May be requested if the physician is certain that the patient has a certain clotting factor deficiency
Factor Substitution Test/Mixing Studies
Match the following.
- Lacks no clotting factors
- Lacks II, VII, IX, and X (Vitamin K dependent factors)
- Lacks I, V, VIII, X, and 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
- Has 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 are the four (4) correction reagents used in mixing studies?
- Normal aged plasma
- Aged plasma
- Adsorbed plasma
- Aged serum
This correction reagent is freshly collected and should not stand for more than 4 hours.
Normal fresh plasma
Assesses the extrinsic and common pathway
PT
Asseses the intrinsic and common pathway
APTT
Match the following.
- Factor I, II, V, X, and XIII
- Factor VIII, IX, XI, XII, PK and HMWK
- Factor III and VII
A. Extrinsic Pathway
B. Intrinsic Pathway
C. Common Pathway
- C
- B
- A
Determiantion of Deficient Clotting Factors. Fill in the blanks.
- First, determine what factors are assessed by ____ and ____.
- Next, analyze the result of the substitution test which adds the correction reagents: ____ and ____.
- To determine the factor that is present in the aged plasma, which corrects the APTT, but not in the adsorbed plasma, the ____ will be performed.
- Following this is determining the pathway the factors are under – extrinsic, intrinsic, or common. By knowing these, the specific factor that the patient is deficient of will be determined.
- First, determine what factors are assessed by APTT and PT.
- Next, analyze the result of the substitution test which adds the correction reagents: adsorbed plasma and aged serum.
- To determine the factor that is present in the aged plasma, which corrects the APTT, but not in the adsorbed plasma, the cancel out technique will be performed.
- Following this is determining the pathway the factors are under – extrinsic, intrinsic, or common. By knowing these, the specific factor that the patient is deficient of will be determined.
Which factor is deficient?
APTT: Prolonged
PT: Normal
Adsorbed Plasma: APTT not corrected
Aged Serum: APTT corrected
Factor IX
Which factor is deficient?
APTT: Prolonged
PT: Normal
Adsorbed Plasma: APTT corrected
Aged Serum: APTT not corrected
Factor VIII
Which factor is deficient?
APTT: Prolonged
PT: Prolonged
Adsorbed Plasma: APTT & PT not corrected
Aged Serum: APTT & PT corrected
Factor X
Which factor is deficient?
APTT: Prolonged
PT: Prolonged
Thrombin Time: Prolonged
Adsorbed Plasma: APTT & PT corrected
Aged Serum: APTT & PT not corrected
Factor I
Which factor is deficient?
APTT: Normal
PT: Prolonged
Adsorbed Plasma: PT not corrected
Aged Serum: PT corrected
Factor VII
- If there is no clotting factor deficiency, this may cause abnormality in PT and APTT results.
- If the test is not being corrected by the correction reagents, it is not due to a certain factor deficiency.
- It is most likely due to a certain inhibitor or circulating anticoagulant.
Inhibitor Studies
Circulating in the plasma which occur when antibodies produced against specific components as a result of replacement therapy in patients with factor deficiencies.
Inhibitors/Anticoagulants
What are the two (2) categories of inhibitor studies?
- Specific
- Non-specific
Categories of Inhibitor Studies
- Directed against specific coagulation factors
- Usually associated with bleeding
Specific Inhibitor Studies
Categories of Inhibitor Studies
- Not directed against specific coagulation factors
- Not associated with bleeding
- Lupus Anticoagulant: Most common type (against phospholipids)
Non-specific Inhibitor Studies
What does it mean when you add normal fresh plasma and the results of PT/PTT are corrected?
Factor Deficiency
What does it mean when you add normal fresh plasma and the results of PT/PTT are ** not corrected or not changed?**
Indicates the presence of a circulating inhibitor
What are the two (2) steps that may be taken to identify the circulating inhibitor?
- Identification of Lupus Anticoagulant
- Specific Factor Inhibition Assay
- Factor VIII inhibitors may be quantitated by mixing varying dilutions of patient plasma with normal pool plasma (NPP), containing a known amount of Factor VIII.
- Factor VIII levels are then measured in all tubes (patient dilutions and NPP).
- The activity of Factor VIII inhibitor in the normal pooled plasma of the mixture will be inhibited.
Factor VIII Inhibitor Assay (Bethesda Method)
May be quantitated by mixing varying dilutions of patient plasma with normal pool plasma (NPP), containing a known amount of Factor VIII.
Factor VIII inhibitors
Mixtures for the Factor VIII Inhibitors Assay are usually incubated at what temperature for how long?
37 deg C for 2 hours
- The FVIII activity that was not destroyed and was not neutralized by the inhibitor added.
- Determined by comparing the difference between the factor activity of the patient and the normal pool plasma mixture with that of your normal pool plasma.
- The so-called Bethesda units
Residual Factor VIII Activity
The reciprocal of the dilution that caused the neutralization of 50% of Factor VIII in normal plasma.
One Bethesda unit
Match the following.
- ≤5 Bethesda units and titers don’t increase following FVIII administration.
- Treatment: Activated Prothrombin Complex (aPCC) like FEIBA – Factor VIII inhibitor bypassing activity, plus steroid or immunomodulation therapy
- Treatment: Raised Factor VIII concentrate dose to increase level.
- > 5 Bethesda units and titer increases following FVIII administration.
A. Low Responders
B. High Responders
- A
- B
- A
- B
Study the procedure for the Factor VIII Inhibitor Assay and the Clinical Diagnostic Test to Differentiate Hemophilia to vWF Disease.
Go niyo na.
In which parameters are hemophilia A and vWF diseases have the same results?
Clue: There are four (4).
- Clot Retraction Time
- Platelet Count
- PT
- APTT
- Assuming that you’ve identified that there is a certain factor deficiency then the next thing that you will do now is to assess the concentration of that particular factor deficient.
- Normal plasma concentration of each of these factors is in the range of 50 to 150% activity.
Factor V (II, VII, X) Assay
Used to determine the plasma concentration of Factors II, V, VII, and X.
Prothrombin Time (PT)
What are the three (3) reagents used in Factor V (II, VII, X) Assay?
- Factor V deficient substrate
- Reference Plasma
- Normal and abnormal control plasma assayed for factor V
Factor V (II, VII, X) Assay
What is performed on the Factor V deficient substrate?
Prothrombin Time
Factor V (II, VII, X) Assay
T or F: Patients with factor 5 deficiency containing the different dilution of patient plasma will be used to correct the abnormality of PT.
T
Factor V (II, VII, X) Assay
The factor 5 content of the patient’s plasma is usually expressed as?
The percentage of normal
Study the dilutions for factor assays.
Go niyo na.
The tests for factor assays are usually done using?
Fibrometer
If you want to detect if the patient is suspected of having the inhibitor called lupus anticoagulant.
Tests for Lupus Anticoagulant
What are the five (5) tests used for Lupus Anticoagulant?
- Platelet Neutralization Procedures (PNP)
- Dilute Russell Viper Venom Time (DRVVT)
- Silica-Based PTT
- Kaolin Clotting Time (KCT)
- Dilute Thromboplastin Time (DTT)
What are the guidelines for lupus anticoagulant detection?
- Prolong phospholipid-dependent clot formation using screening assay such as low phospholipid PTT or DRVVT.
- Failure to correct clotting when mixing with normal plasma.
- Shortening or complete correction of the prolonged screening assay by employing addition of excess phospholipid.
- Exclusion of another anticoagulant.
- One of a group of tests for the detection of lupus anticoagulants.
- An increase in the amount of phospholipid is added usually to the test system in order to minimize the effect of phospholipid-dependent anticoagulant, in which then it will produce a shortened clotting time.
Platelet Neutralization Procedures (PNP)
- When you mix the platelet poor plasma with the suspension of the ruptured platelets — which will serve as a source of phospholipids — plus APTT reagent and calcium chloride, you will come up with the clotting time result.
- The clotting time is noted and compared with the clotting time of a similar mixture, which is usually what you substituted in sodium chloride (NaCl) for the platelets.
Freeze-Thawed Platelet Suspension Neutralized LA
T or F: If a lupus inhibitor is present, then the effect of the anticoagulant will be increased or it will be bypassed by the freeze-thawed platelets.
F (decreased)
Match the following.
- < 8 seconds
- > /= 8 seconds
A. Negative
B. Positive
- B
- A
The major type of this lupus anticoagulant
Cardiolipin antibody
- Either IgG, IgM, or IgA, and may also be a mixture of the antibodies that inhibit the in-vitro assembly of the prothrombinase complex.
- Inhibit the activation of prothrombin, that’s why it will interfere with the phospholipid-dependent clotting factors, such as PT, APTT, and DRVVT.
Lupus Anticoagulant
A single vial test reagent containing the
Russell Viper Venom.
DRVVT
- Russell Viper Venom, Factor V, Phospholipid, and Calcium ions (Prothrombin Activators) will activate ____
* After activating factor X, it will now begin the coagulation system, which is the conversion of ____
* When this reagent is added to the plasma containing the LA, some of the phospholipids in the test system will be neutralized by LA.
* As a result, it will limit the amount of phospholipid available for coagulation, resulting in ____ of clotting time, compared with a normal plasma.
- Russell Viper Venom, Factor V, Phospholipid, and Calcium ions (Prothrombin Activators) will activate Factor X.
* After activating factor X, it will now begin the coagulation system, which is the conversion of prothrombin to thrombin.
* When this reagent is added to the plasma containing the LA, some of the phospholipids in the test system will be neutralized by LA.
* As a result, it will limit the amount of phospholipid available for coagulation, resulting in prolongation of clotting time, compared with a normal plasma.
Please study Algorithm for Investigation of Abnormal Coagulation Tests.
Go niyo na, ang haba e pota.
- Principle: Whole blood will clot spontaneously when clotted in a glass tube without anticoagulant.
- Result: The clot should remain INTACT for approximately 48 hrs. (2 days) at 37C.
- If there is an excess systemic fibrinolysis, then the clot lysis or dissolution will occur prior to 48 hrs. The rate should be normal.
Whole Blood Clot Lysis Time
- This test is used to detect increased fibrinolysis.
- It is only able to detect high increase in fibrinolytic
activity.
Whole Blood Clot Lysis Time
- Avoids the problems of plasminogen inhibitors in the assay system. In this test, the result is more rapid and sensitive assay of lytic activity.
- **Principle: ** Addition of 1% acetic acid to diluted plasma causes the euglobulin portion of the plasma to precipitate.
- After precipitating, you will centrifuge the tube and remove the supernatant, the euglobulins are dissolved in a buffer solution.
- After the supernatant is removed, thrombin is added to the tube and incubated at 37 C.
Euglobulin Clot Lysis Time
Proteins that precipitate when plasma is diluted with water and acidified.
Euglobulin
Lysis in less than 2 hours is indicative of?
Increased fibrinolytic activity
What are the two (2) types of Lysis Products Determination?
- Protamine Sulfate Dilution Test
- Ethanol Gelation Test
- Detects presence of fibrin monomers of FDPs by causing fibrin strands or “gel-like” clots (PARACOAGULATION)
- NORMAL RESULT: NO GEL FORMATION indicates the presence of fibrinolysis.
Protamine Sulfate Dilution Test
- Less sensitive but more specific than Protamine Sulfate Dilution Test
- PRINCIPLE: Fibrin soluble monomers in the presence of 50% ethanol solution will dissociate resulting in its polymerization and subsequent
- NORMAL: NO GEL FORMATION
Ethanol Gelation Test
- Usually performed with the aid of latex particles coated with antibody against fibrin or fibrinogen fragments D and E are mixed with patient’s plasma
- POSITIVE RESULT: presence of macroscopic agglutination indicating presence of FDPs
Latex FDP Test/Fibrin Degradation Product
- Measures clot specific fragment arising from degradation of cross-linked fibrin (D-dimer or the latex D-dimer) and not fragment X, Y, D, and E
- Positive result is a specific evidence of INTRAVASCULAR FIBRIN FORMATION
- Positive in DIC, Pulmonary/Central Embolism, and
Thrombosis
Latex D-dimer Assay
T or F: Plasminogen deficiencies may be inherited or acquired.
T
Plasminogen is (increased/decreased) in thrombotic therapy, fibrinolytic disorders, liver disease, and DIC.
Decreased
- Measured by electroimmunoassay and radioimmunoassay (RIA).
- Functional assays include fibrin plate and chromogenic assays.
Plasminogen Assays
Principle of Plasminogen Assay
- Patient plasma is incubated with an excess of ____ reagent.
- Plasminogen present in the plasma specimen forms a ____ which possess plasmin-like activity.
- When a substrate is added, the plasma-streptokinase
mixture, this plasmin-like activity will release paranitroanilide (pNA). - ____ will release from the substrate. It is the one being measured in spectrophotometry and the value of this product is directly proportional to the amount of plasminogen present in your plasma
- The concentration of your patient plasminogen is usually read from a reference curve.
- Patient plasma is incubated with an excess of STREPTOKINASE reagent.
- Plasminogen present in the plasma specimen forms a PLASMINOGEN-STREPTOKINASE COMPLEX which possess plasmin-like activity.
- When a substrate is added, the plasma-streptokinase
mixture, this plasmin-like activity will release paranitroanilide (pNA). - Paranitroanilide (pNA) will release from the substrate. It is the one being measured in spectrophotometry and the value of this product is directly proportional to the amount of plasminogen present in your plasma
- The concentration of your patient plasminogen is usually read from a reference curve.
- Plasma is incubated with the protein C activator (lyophilized venom enzyme from Agkistrodon contortrix, which is known as a copperhead snake).
- Upon the addition of the substrate, activated protein C will catalyze the release of paranitroanilide (pNA) from the substrate
- The amount of pNA released is measured by spectrophotometer; it is DIRECTLY PROPORTIONAL to the concentration of protein C in the plasma.
- When adding certain reagents, like 20% acetic acid, it will stop the reaction. Therefore, it is imperative that the tube be mixed well as soon as the acetic acid is added. Once added, the color will remain stable for 4 hours.
Protein C Chromogenic Method
- Diluted plasma is incubated with protein C deficient plasma in the presence of protein C activator
- The amount of protein seen in plasma is related to the degree of correction obtained when the diluted plasma is incubated with protein C deficient plasma in the presence of the protein C activator.
- The activated protein C inhibits Factor V and VIII (labile
factor), thus will cause prolonged APTT. - The clotting time obtained for each plasma dilution is
INVERSELY PROPORTIONAL to the percentage of protein C activity.
Protein C Clotting Assay
- Plasma dilutions containing protein S antigen are incubated in microwells coated with anti-protein S antibody
- Anti-protein S peroxidase conjugate is added, in which it will bind to the free antigenic determinants of protein S, forming a sandwich
Protein S
- Protein S bound to C4b BP is removed from the plasma by precipitating with polyethylene glycol.
- Protein S, which is a Vitamin K-dependent protein is
available in two forms.
Free Protein S
Match the following.
- 60%
- 40%
A. Protein S Antigen
B. Free Protein S
- May be acquired or inherited.
- Typically, it is associated with an increased risk for recurrent venous thrombosis.
Protein S Deficiency
- Principle: The percentage of the functional Protein S present in the plasma is usually determined by the prolongation of the clotting time obtained.
- Diluted plasma is incubated with Protein S deficient plasma in the presence of activated protein C and Factor Va.
Protein S Clotting Assay
Disclaimer: Please read more into the details ng lahat ng ‘to, it’s kinda lengthy so please just review to be sure.
Oki bye sorry this deck was late </3
