MT UNIT 6.4: Mixing Studies and Factor Substitution Assays Ms.

Diana Mendoza LAB

2022 MT6325: Hematology 2 2nd Shifting | Medical Technology | University of Santo Tomas

OUTLINE COAGULATION PATHWAY

Mixing Studies 1
Coagulation Pathway
- Extrinsic Pathway
- Common Pathway
- Coagulation Factors Active Form and Pathway Participation 2
Factors that Prolong PT and PTT/aPTT
- Factors that Prolong PT
- Factors that Prolong PTT/aPTT
- PT vs PTT/aPTT
- PT vs PTT/aPTT vs TT
Factor Substitution Assay Mixing Studies
- Correction Reagents
- Interpretation of Results
Mixing Studies Laboratory Procedure 3
- Materials Coagulation Pathway
- Preparation of Specimen
Lupus Anticoagulant (LAC)
Exercise
- Explanation of examples
- Clinical Significance of Specific Factor Deficiency
LEGEND
BLACK TEXT COLORED TEXT
Based from ppt Based from lecture proper

MIXING STUDIES Extrinsic Pathway

● In a serum or plasma, we do not know the cause of prolonged
Prothrombin Time (PT) or Partial Thromboplastin Time (PTT).

Extrinsic Pathway
- Factor III reacts extrinsically to convert factor 7 to 7a
- 7a would then enter the common pathway.

Centrifuged blood
- In PT and PTT determination, we use plasma using a light blue
top.
- The plasma will be subjected to coagulation studies.
- If a patient has prolonged PT or aPTT (Artificial Partial Common Pathway
Thromboplastin Time), there is something with the plasma that
needs to be determined.
● Mixing studies are performed to identify specific factor deficiency
and to rule out or rule in the presence of inhibitors.
○ Done by mixing correction reagent with the patient's plasma
and performing PT and/or PTT again.
○ We could see if the PT and PTT is corrected or not corrected.
● Abnormal coagulation test results may be due to:
○ Specific missing factor
○ Presence of inhibitors or anticoagulants
■ Lupus anticoagulants
→ Acts as anticoagulant which prolongs PT and PTT
■ Antibodies against the coagulation factors (i.e., factor VIII)
■ Heparin-like activities
■ Antithromboplastin Common Pathway
- Common Pathway involves Factor X, V, II and I
■ Autoantibodies
- Once factor X is activated, it reacts with platelet factor III,
● Factor Substitution Assay
factor V and calcium.
○ Done to further identify the immediate cause of elevated
- Needed by prothrombin (factor II) to become thrombin
coagulation results.
(factor IIa)
- Thrombin: convert fibrinogen (factor I) to fibrin (factor
Ia)

AUTENCIO, Rojiellen Angela | 3I-MT Hustle kahit Hassle 💯 1

 MT UNIT 6.4: Mixing Studies and Factor Substitution Assays Ms. Diana Mendoza LAB
2022 MT6325: Hematology 2 2nd Shifting | Medical Technology | University of Santo Tomas

Coagulation Factors, Active Form and Pathway Participation PT vs PTT/aPTT vs TT (Thrombin Time)
Coagulation Active Form Pathway Participation Deficient Factors PT PTT/aPTT TT
Factors I Abn Abn Abn
I Fibrin Clot COMMON II Abn Abn N
V Abn Abn N
II Serine Protease COMMON
VII Abn N N
V Cofactor COMMON VIII:C N Abn N
VII Serine Protease EXTRINSIC IX N Abn N
VIII:C Cofactor INTRINSIC X Abn Abn N
XI N Abn N
IX Serine Protease INTRINSIC
XII N Abn N
X Serine Protease COMMON Abn: Abnormal; N: Normal
XI Serine Protease INTRINSIC FACTOR SUBSTITUTION ASSAY MIXING STUDIES
● Uses serum and plasma.
XII Serine Protease INTRINSIC
● Done by adding aged plasma, adsorbed plasma, and serum to
● Factor I abnormal plasma to determine if the results will be corrected.
○ Inactive form: Fibrinogen ● End point = correction of PT, aPTT, TT results
○ Active form: Fibrin clot (fibrin monomer/polymer) ● Not routinely done anymore due to the following reasons:
● Factor II ○ Equivocal results at time
○ Needed in conversion of fibrinogen to become activated fibrin ○ Most of the congenital deficiencies are either VIII, IX or XI
○ Availability of the one-staged clot-based factor assays
FACTORS THAT PROLONG PT AND PTT/aPTT
● Partial Thromboplastin Time (PTT) or aPTT (Artificial Partial Mixing Correction Reagents
Thromboplastin Time) ● Mixed together with the patient sample and we later on identify if the
○ Monitor heparin therapy PT or PTT are corrected or not.
○ Detects coagulation factor deficiency in intrinsic and common ● Used as a basis to identify the missing factors.
pathways.
● Prothrombin Time (PT) Fresh/Normal Plasma
○ Monitor Coumadin or warfarin ● Contains all coagulation factors.
○ Detects coagulation factor deficiency in extrinsic and common
pathways. Aged Plasma
● Commonality between the two tests is that they are both prolonged
● Known to kill labile factors.
by common pathway factors.
● Does not contain labile cofactors V and VIII.
○ Factors: X, V, II, I
Adsorbed plasma
Factors that prolongs PT
● Does not contain Vitamin K-dependent factors (II,VII, IX, X).
● Both extrinsic and common pathway factors prolongs PT.
● Prolonged PT and a normal PTT:
Aged Serum
○ Only involve factors in the extrinsic pathway.
■ Factors III & VII ● Does not contain factors V, VIII, XIII (stabilizing factor), fibrinogen
(factor I) and prothrombin (factor II).
Factors that prolongs PTT or aPTT ● The basis of the differentiation of plasma and serum is that serum
● Both intrinsic and common pathway factors prolongs PT. does not contain fibrinogen.
● Prolonged PTT/aPTT and a normal PT: ○ In a normal blood collection, it is inhibited by preventing the
○ Only involves factors in the intrinsic pathway. action of thrombin.
■ Factors XII, XI, IX and VIII
Presence/Absence of Factors Based on Mixing Correction Reagents
PT vs PTT/aPTT Coagulation Fresh Aged Adsorbed Fresh Aged
Factors Plasma Plasma Plasma Serum Serum
PT PTT or aPTT
I ✓ ✓ ✓ X X
Therapy it monitors Coumadin or Monitor heparin
warfarin therapy II ✓ ✓ X X X
Detects deficiency in what Extrinsic and Intrinsic and V ✓ X ✓ X X
pathway common pathway common pathway
VII ✓ ✓ X ✓ ✓
Factors involved in prolonged III, VII XII, XI, IX, VIII
results (except common VIII ✓ X ✓ X X
pathway factors) IX ✓ ✓ X ✓ ✓
X ✓ ✓ X ✓ ✓
XI ✓ ✓ ✓ ✓ ✓
XII ✓ ✓ ✓ ✓ ✓
XIII ✓ ✓ ✓ X X
✓: present; X: absent

AUTENCIO, Rojiellen Angela | 3I-MT Hustle kahit Hassle 💯 2

 MT UNIT 6.4: Mixing Studies and Factor Substitution Assays Ms. Diana Mendoza LAB
2022 MT6325: Hematology 2 2nd Shifting | Medical Technology | University of Santo Tomas

Interpretation of Results
DEFICIENT FACTORS PT APTT TT SUBSTITUTION STUDIES
NORMAL PLASMA ADSORBED PLASMA AGED SERUM
I Abn Abn Abn C C NC
II Abn Abn N C NC NC
V Abn Abn N C C NC
VII Abn N N C NC C
VIII: C N Abn N C C NC
IX N Abn N C NC C
X Abn Abn N C NC C
XI N Abn N C C C
XII N Abn N C C C

MIXING STUDIES LABORATORY PROCEDURE LUPUS ANTICOAGULANT (LAC)

● Materials: ● LAC will not be corrected in Factor Substitution Assay using normal
○ Two-way needle plasma.
○ Citrated evacuated tube ● Perform LAC-specific testing:
○ Centrifuge ○ I.e. DRVVT- Dilute Russell Viper Venom Test
○ 0.7 mL serological pipette
○ 0.8 mL serological pipette
○ Wasserman tubes
○ 37OC water bath
○ aPTT reagent
○ CaCl2
○ Timer
○ Tissue paper

Preparation of Specimen
1. Collect venous blood in a citrated tube.
2. Centrifuge specimen at 1,500 rpm.
3. Separate immediately the plasma in a separate clean dry test Lupus Anticoagulant (LAC)
tube.
4. Test should be done 2 hours after collection. EXERCISE
● Patient Plasma ● Determine the missing factor based on the given
○ Tube 1 = 9 parts Given
○ Tube 2 = 5 parts PT aPTT Fresh Adsorbed Aged Aged Deficient
○ Tube 3 = 1 parts Plasma Plasma Serum Plasma
● Normal Plasma Normal ↑ Corr. Corr. Not. Corr. Not. Corr. VIII
○ Tube 1 = 1 parts ↑ ↑ Corr. Corr. Not. Corr. Not. Corr. V
○ Tube 2 = 5 parts
↑ ↑ Not. Corr. ?. ? ? Indef
○ Tube 3 = 9 parts
Normal ↑ Corr. Not. Corr. Corr. IX
What if there’s no correction of results after factor substitution ↑ ↑ Not. Corr Not. Corr II
assays using serum or plasma? ↑: Prolonged; Corr: Corrected; Not Corr: Not Corrected; Indef: Indefinite
● Possible presence of inhibitors and antibodies against the
coagulation system. Normal PT; Prolonged aPTT
○ Lupus anticoagulants ● Given that the aPTT is prolonged and known to detect deficiency in
○ Antibodies against the coagulation factors (i.e. Factor VIII) the intrinsic and common pathway.
○ Heparin-like activities ● In this case, PT is normal so we do not consider the common
○ Antithromboplastin pathway.
○ Autoantibodies ● Choices include: XII, XI, IX and VIII
○ After adding fresh plasma aPTT is corrected, which indicates
Anticoagulants that the problem in this patient is deficient in coagulation
factor.
■ Fresh plasma has all the coagulation factors which
corrected the missing one.
○ After adding adsorbed plasma it is corrected
■ Adsorbed plasma does not have factor II, VII, IX and X.
■ There is something in the factors contained in the
adsorbed plasma that corrected the aPTT.
■ Cross out factor IX: adsorbed plasma does not have this
factor but it was still corrected.
○ After adding aged serum it is not corrected
■ Aged serum does not have factor I, II, V, VIII, XIII.
■ Cross out factor XII and XI: it is contained in the aged
serum hence we do not consider it as a missing factor
AUTENCIO, Rojiellen Angela | 3I-MT Hustle kahit Hassle 💯 3
 MT UNIT 6.4: Mixing Studies and Factor Substitution Assays Ms. Diana Mendoza LAB
2022 MT6325: Hematology 2 2nd Shifting | Medical Technology | University of Santo Tomas

■ Factor VIII Deficient

→ Clinical significance: Hemophilia A

Prolonged PT; Prolonged aPTT

● Since both are prolonged, it involves the common pathway.
● Choices include: X, V, II, I
○ Corrected fresh plasma
■ Deficiency in coagulation factor
○ Adsorbed plasma corrected
■ Does not include X, VII, IX and II
■ Cross out X and II
○ Not corrected with the aged serum
■ Not helpful to rule out factor V and I as this correction
reagent does not contain the said factor also that is why it
is definitely not corrected.
○ Not corrected with aged plasma
■ No factors V and VIII
■ Factor V deficiency

Prolonged PT; Prolonged aPTT

● Fresh plasma is not corrected
○ Do not proceed with the analysis of other correcting reagents.
○ Possible presence of circulating anticoagulants.

Normal PT; Prolonged aPTT

● Involves intrinsic pathway factor deficiency
● Choices: XII, XI, IX and VIII
○ Corrected fresh plasma
■ Factor Deficiency
○ Adsorbed plasma not corrected
■ No II, VII, IX and X
■ Factor IX Deficiency
○ Check aged serum to confirm
■ No I, V, VIII, XIII, II
■ Rule out VIII
■ Rule out XII and XI: contained in aged serum and it is
corrected

Prolonged PT; Prolonged aPTT

● Involves the common pathway.
● Choices include: X, V, II, I
○ Adsorbed plasma not corrected
■ No factors II, VII, IX, X
■ Rule out V and I
■ Factors II and X remain
○ Aged serum not corrected
■ No factors I, V, VIII, XIII, II
■ Factor II deficiency

Clinical Significance of Specific Factor Deficiency

DEFICIENT FACTOR CLINICAL SIGNIFICANCE
V Owren’s disease/Parahemophilia
VIII Classic Hemophilia/Hemophilia A
XI Rosenthal Syndrome/Hemophilia C
XIII Analyzed using 5M Urea Clot Solubility Test to
validate Factor XIII Deficiency

AUTENCIO, Rojiellen Angela | 3I-MT Hustle kahit Hassle 💯 4