MODULE 5: CLOT-BASED SCREENING TESTS

HEMATOLOGY 2 LABORATORY
MLS 12b ll MAY 2022
Transcribers: Almario, Altura, Gallego, Jovero, Saradolla
University of San Agustin- Iloilo
CLOT-BASED SCREENING TESTS
LEGEND (TESTS FOR SECONDARY HEMOSTASIS):
Book Neo recorded Lab Lecturer Neo Highlights ● Measures the clot formation
vid PPT A1 recorded vid
Orange Pink Blue Violet Red CLOTTING TIME
● Measures in vitro coagulation, especially in intrinsic
OUTLINE pathway
Tests for Secondary Hemostasis: Clot-Based Screening
Tests 3 Methods:
1. Capillary Method
Clotting Time 1 ○ Evaluate the extrinsic pathway of coagulation
● Capillary Method 1 ○ Applicable only for infants and patients receiving
● Drop/Slide Method 2 heparin therapy
● Lee White Clotting Time 2
Prothrombin Time 3 2. Drop/Slide Method
○ Extrinsic Pathway: A Review 3 ○ Evaluate the intrinsic pathway of coagulation
● Principle and Methods of PT 4
○ Quick Method (Manual Method) 5 3. Lee-White Clotting Time
○ Prothrombin Time (Diacheck C1) / One 5 ○ Assess in vitro coagulation
Stage PT
● Reporting of Results and Sources of Error 6
Principle
● Diagnostic Importance 7
Activated Partial Thromboplastin Time 7 ● Clotting time of whole blood is the length of time required to
○ Review: Major Components of 7 form a clot in vitro under standard conditions.
Hemostasis
● aPTT Methods and Procedures 11 ● In this test, whole blood will form a solid clot when exposed
○ Sample Requirements 11 to a foreign surface such as a glass tube/glass slide.
○ Test Reagent 11 ● It is also the time interval from puncture to form fibrin
○ Test Procedure 11 strands. This test is used to diagnose and assess bleeding
● Sample of Manual and Semi-Automated Method 11 problems and monitor anticoagulant therapy.
○ Manual Method 11
○ Semi-automated Method 11 CAPILLARY METHOD
○ Fully Automated Machine 12 ● Applicable only to infants and patients receiving heparin
○ aPTT using Diacheck Machine 12 therapy.
● Standard Reference Value and Sources of Error 12 ● evaluates the extrinsic pathway of coagulation
● Diagnostic Importance 12
○ Pathology 12 Materials Needed
■ DIC 13
● 70% alcohol
■ Hemophilia 13
● Lancet
■ Rosenthal Syndrome 13
● Stopwatch
■ Vit. K Deficiency 13
● Cotton
■ Acquired Factor Inhibitor 13
● Non-anticoagulated capillary tube (Blue tipped)
■ Advanced Liver Diseases 13
■ Anticoagulant Medications 14
■ Lupus Anticoagulants 14 Procedure
Thrombin Clotting Time 14 1. Prepares the materials required for the test.
● Clinical Significance: Prolonged TCT 14 2. Identifies the patient prior to collection.
Mixing and Substitution Studies 15 3. Explains the procedure.
● Preparation of Blood Derivatives 15 4. Selects and warms the puncture site (3rd or 4th finger)
○ Summary of Factors Found in Blood 15 5. Cleanses the incision site. Allows alcohol to dry.
Derivatives 15 6. Puncture the site.
● Mixing Studies 15 7. Wipe off the first drop of blood.
○ Indicators 15 ● To prevent the contamination of the blood
● Substitution Studies 15 sample with tissue fluid, as it is rich in tissue
Venom Activated Assays 17 factors that may activate the extrinsic pathway of
● Reptilase Time 17 coagulation.
● Russell's Viper Venom Time 17 ○ Tissue fluid → initiate clotting
● To prevent the residual alcohol
● To facilitate the free flow of blood
Lab A2 Intro 8. Start the timer as soon as the second drop of blood
PRIMARY SECONDARY appears.
HEMOSTASIS HEMOSTASIS 9. Fills a plain capillary tube with blood.
10. Breaks off a portion of the tube every 30 seconds.
Composed of: Platelet, capillaries Platelet, Coagulation
11. Stop the timer as soon as fibrin strands are seen bridging
Factors
the two broken ends of the tube.
Tests: Bleeding Time Clotting Time
12. Applies after care.
↓ 13. Record clotting time accurately.
PT, APTT, and TCT 14. Identifies and segregates wastes generated and disposes
↓ them properly.
Mixing Studies or
Substitution Studies Reference Value
3 - 7 minutes

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[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
Procedure
1. Adhere to safety precaution by using PPE.
2. Prepare the materials required for the test.
3. Identify the patient prior to collection.
4. Explain the procedure.
5. Select and warm the puncture site.
● (3rd or 4th finger)
6. Cleanses the incision site. Allow alcohol to dry.
7. Punctures the site without applying pressure.
8. Wipe off the first drop of blood.
Figure: Capillary Method
9. Starts the timer.
10. Obtain 3 drops of blood consecutively in a clean dry glass
slide not touching the skin.
● Label the glass slide: 1 → 2 → 3 and place a drop
in each labeled no.
● Checking clot formation : 3 → 2 → 1
11. Using a lancet or needle, check fibrin thread formation
starting at the last drop of blood every 30 seconds. Move to
the second drop once the fibrin is seen on the last
● Fibrin thread formation checking:
○ Drop 3 → 2 → 1
12. Stop the timer as soon as a fibrin strand has formed on the
Capillary Tube first drop.
● Every 30 sec, a portion is broken off 13. Applies after care.
○ Every broken portion represents 30 sec 14. Records clotting time accurately.
● Endpoint: 15. Identifies and segregates wastes generated and disposes
○ Broken until fibrin strands are seen bridging them properly.

Reference Value
2 - 6 minutes

Figure: Fibrin strand bridging the two broken ends of the capillary
tube

Sources of Error
1. Failure to wipe off the first drop of blood.
● This may be contaminated by tissue factor Figure: Fibrin Thread Formation
through pricking and may yield to shortened
clotting time.
2. Underfilled capillary tube Sources of Error
● Should be filled until ¾ of its length 1. Failure to wipe off the first drop of blood.
● Shortened clotting ● This may be contaminated by tissue factor
○ Less volume and anticoagulated tube through pricking and may yield to shortened
3. Failure to keep track of time clotting time.
● Must be every 30 sec. 2. Contact of the slide to the puncture site
4. Skin thickness at the puncture site ● To avoid premature activation of contact factors
● Avoid heavily calloused areas ● Shortened clotting time
3. Failure to keep track of time
4. Drying of the blood sample on the slide
DROP or SLIDE METHOD
● Can not observe the fibrin strand formation
● used to evaluate the intrinsic pathway of coagulation which
involves Factors VIII, IX, X, XI and XII
○ Factor X is from common pathway LEE AND WHITE COAGULATION TIME
● Lee-White coagulation time test was first described in 1913.
Recap (Lecture, Module 5) ● This test was the first laboratory procedure to assess
coagulation in vitro.
STAGES OF COAGULATION PATHWAY ● Also known as whole blood coagulation time or venous
1. Generation of thromboplastin coagulation time.
● Extrinsic: III - VII ○ Whole blood, when removed from the vascular
● Intrinsic: XII -XI - IX - VIII system and exposed to a foreign surface, will
● Common: X – V – II - I form a solid clot.
2. Conversion of prothrombin to thrombin
3. Conversion of fibrinogen to fibrin clot Principle
● Glass tube is used to initiate the clotting formation
Materials Needed ● The time interval from the initiation of clotting to visible clot
formation reflects the condition of coagulation mechanism.
● 70% alcohol ● The whole blood clotting time is a rough measure of all
● Lancet intrinsic clotting factors in the absence of tissue factors.
● Stopwatch ● Variations are wide and the test sensitivity is limited.
● Cotton ● Within limits, the time required for the formation of the solid
● Slides clot is a measure of the coagulation system.

Materials Needed

Venipuncture Materials LWCT Materials

1. 70% alcohol 1. Plain test tubes with label
2. Tourniquet (Tube 1, Tube 2, Tube 3)
3. 5 cc Syringe 2. Water Bath set at 37ºC
4. Cotton & Plaster 3. Timer/Stopwatch

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[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
Procedure PROTHROMBIN TIME
1. Adheres to the safety precautions by using PPE. ● One of the clot-based assays to detect factor deficiencies
2. Prepares the materials required for the test. ● Basically focuses on checking if an individual has a
3. Identifies the patient prior to collection. deficiency, specifically in extrinsic factors of the extrinsic
4. Explains the procedure. pathway of coagulation
5. Label the glass tubes #1, #2 and #3 and place them in a ○ In contrast to aPTT, another clot-based assay
water bath at 37ºC that focuses mostly on the intrinsic factors of the
● At 37ºC, it will be delivered while it mimics the coagulation pathway
body temperature, in vivo ● Measures extrinsic and common pathway
6. Cleanses the puncture site. Allows the alcohol to dry. ● Also known as PT, is one of the assays to screen for
7. Withdraws 4 ml of blood coagulation factor deficiencies in the extrinsic pathway.
8. Start the timer as soon as blood enters the syringe. ● Detects the absence or deficiency of coagulation factors in
9. Delivers 1 ml of blood in each tube starting with tube 3, the the extrinsic pathway.
tube 2, and tube 1. ● It is usually performed together with Activated Partial
● Checking clot formation : 1 → 2 → 3 Thromboplastin Time or APTT
10. Discards the last ml of blood. ○ focuses on the detection of intrinsic pathway
● LWCT is a rough measure of all intrinsic clotting deficiencies
factors in the absence of tissue factors. ○ as PT cannot detect the deficiency in intrinsic
● Discarded because the 1st portion of blood has pathway
come in contact or contaminated with tissue ○ done together to compare which pathway is
factor affected
● Last ml is the first portion of blood that entered ○ If PT is prolonged → problem in extrinsic
the syringe → there can already be an initiation pathway;
of clotting ■ If aPTT is prolonged: intrinsic pathway
11. Replaces the tubes in the water bath after each delivery.
12. Checks for clot formation starting at tube 1 after exactly 5 The most common use of PT, however, is to monitor the effects of
minutes. Coumadin,
13. Applies after care. ● Warfarin, coumarin
14. Records clotting time accurately. ○ Vitamin K antagonist
15. Identifies and segregates waste generated and disposes ○ an oral anticoagulant being used by patients
them properly. undergoing anticoagulant therapy.
● Mostly with high risk for
thrombosis
● Coumadin therapy suppresses FVII, FX, and prothrombin
(FII) production, and thus, prolongs PT.
● Important to monitor the dose
○ Prolonged PT → prone to hemorrhage

PT is… Deficiencies of
Most sensitive Factor VII
● has the shortest lifespan in the
circulation
Moderately sensitive Factor V
Figure: Sample set-up of LWCT Factor X
Sensitive Fibrinogen
● Dispense 1 mL of blood per tube Prothrombin
○ Discard the last mL of blood from the syringe Not sensitive Factor VIII
● Every 5 minutes, check for clot: Tube 1 - 2 - 3 Factor IX
Factor XIII

● These factors are mostly in the intrinsic

pathway
● Factor XIII is a special factor that cannot
be detected either by PT and aPTT and
● Tilt the tube in 1 direction, gently, to check for clot formation has its own test
○ Can only be activated by the
Reference Value presence of thrombin
5 - 15 minutes

Factors Affecting Lee-White Coagulation Time Extrinsic Pathway: A Review

● Factors involved: Tissue factor III and Factor VII
○ Activates common pathway
1. Poor Shortened coagulation time ● Extrinsic pathway is one of the three pathways in the
venipuncture coagulation cascade.
● May promote the release of tissue
technique ● This is called “extrinsic”
factor
○ for this is initiated by the release of Tissue Factor
● There can be a start of coagulation (also known as TF or Factor III) which is found
2. Bubbles entering Shortened coagulation time outside the circulation.
the syringe ■ TF is released from damaged cells
3. Diameter of the Smaller → faster clotting time → ○ the TF will then activate the Factor VII and will
tube Shortened form the complex TF:VIIa in the presence of
Smaller diameter of the tube, more rapid calcium.
conformation may occur ■ this complex can then activate the
4. Temperature Increase of temperature → Shortened Factor IX and Factor X, and
Decrease of temperature → Prolonged ○ the coagulation cascade will continue until the
Temperature is directly proportional formation of the cross-linked fibrin.
● Should be 37ºC, in order to mimic
the body temperature in vivo
○ If not 37ºC, may retard the
coagulation time
5. Admixture of Shortened coagulation time
blood with tissue
juice
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[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
Note:
Factor VII will be activated due to the release of tissue factor due to
injury → Factor VIIa → phospholipid, TF, Factor VII, Ca complexes
→ the complex will activate Factor X → common pathway →
complex: phospholipid,Xa, Va, calcium

Extrinsic pathway, Coagulation factors involved:

(Tip: decreasing order)
● Factor 12
● Factor 11
● Factor 9
● Factor 8

Common pathway Coagulation factors involved:

(Tip: Half by half))
● Factor 10
● Factor 5
● Factor 2
● Factor 1

PRINCIPLE AND METHODS OF PT

Principle of the Test

● When tissue extract or thromboplastin (serve as the TF) is
added to platelet-poor plasma along with calcium, it reacts
with
○ Factor VIIa to convert Factor X to Xa along with
Va, phospholipid and calcium that converts
prothrombin to thrombin.
● The thrombin generated will then convert fibrinogen to
fibrin.
● The time from addition of the thromboplastin-CaCl2 solution
to initiate the formation of the clot
○ reported as the prothrombin time.

● PT reagents, often called thromboplastin or tissue

thromboplastin, consist of recombinant or affinity-purified
tissue factor suspended in phospholipids mixed with a
The coagulation system. Extrinsic pathway in green and blue; buffered 0.025 M solution of calcium chloride
Intrinsic pathway in red and blue; common pathway in blue ● A few thromboplastins are organic extracts of emulsified
rabbit brain or lung suspended in calcium chloride.
Figure 35.10 Plasma-Based Coagulation Cascade. The ● When mixed with citrated PPP, the PT reagent triggers
coagulation cascade consists of the contact system (simplified here) fibrin polymerization by adding calcium ions and activating
and the intrinsic, extrinsic, and common pathways. In the intrinsic plasma factor VII (Figure 41.7)
pathway (red), the contact factors XII, prekallikrein (pre-K), and high- ● Calcium and phospholipids participate in the formation of
molecular-weight kininogen (HMWK) are activated and proceed to the
activate factors XI, IX, VIII, X, and V and prothrombin, which converts ○ tissue factor-factor VIIa complex,
fibrinogen to fibrin. In the extrinsic pathway (green), tissue factor (TF) ○ factor VIIIa-factor IXa complex, and
activates factor VII, which activates factors X, V, and prothrombin, ○ factor Va-factor Xa complex.
cleaving fibrinogen to fibrin. Both the intrinsic and extrinsic pathways ● The clot is detectable by optical or electromechanical
converge with the activation of factor X, so factors X, V, prothrombin, sensors
and fibrinogen are called the common pathway (blue). Dashed boxes ● The PT is prolonged in multiple factor deficiency disorders
indicate the coagulation factor complexes that assemble on that include deficiencies of factors VII and X and is used
phospholipid (yellow symbol). These pathways are the basis of clinical most often to monitor the effects of therapy with the oral
coagulation laboratory tests. Thr, Thrombin. anticoagulant Coumadin

Green: Extrinsic Pathway

Factor VII will be activated by the TF→ Factor VIIa→ presence of Ca

→ complex, can activate both factor X and IX

If Factor X is activated → Factor Xa → presence of Ca → form a

complex with Factor Va → activate the prothrombin (or Factor II) →
generate thrombin → activate fibrinogen → fibrin polymer → stabilized
by Factor XIIIa → Cross-linked fibrin

Note:
Thrombin
● This is goal of the coagulation cascade to generate
thrombin, as this will activate the fibrinogen
○ Convert fibrinogen to a fibrin polymer

Example:
Figure 41.7 Prothrombin time (PT) Assay. The PT reagent
There is a deficiency in one of the factors → Prolonged prothrombin
(thromboplastin) consists of tissue factor (TF), phospholipid (PL), and
time
ionized calcium (Ca2+). The reagent activates the extrinsic and
common pathways of the coagulation mechanism beginning with
factor VII (colored area in figure). The PT is prolonged by deficien-
cies of factors VII, X, and V; prothrombin; and fibrinogen when
fibrinogen is less than 100 mg/dL. The PT is prolonged in Coumadin
therapy because production of factor VII, factor X, and prothrombin is
suppressed. a, Activated form of each factor HMWK, high-molecular-
weight kininogen; Pre-K, prekallikrein; Pro, prothrom- bin (II); Thr,
thrombin.
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[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
Note:Thromboplastin, serve as TF

PRP + TF (reagent)
→ activate Factor VIIa
→ Activate Factor X, together with Factor V
→ convert thrombin to prothrombin
→ convert fibrinogen to fibrin polymer
Figure: Equipment used to dispense samples and reagents in the
From the addition of reagent (thromboplastin) to the formation of the laboratory
clot, it is the Prothrombin Time ● 1st pic: Mechanical pipette
○ Commonly used
Quick Method (Manual Method) ○ Uses disposable tips (2nd pic)
○ Based in uL: adjustable
● 3rd pic: traditional pipette that uses rubber bulb to aspirate
Materials Needed sample
○ Based in mL

Note:
In Extrinsic Pathway, tissue factor is required
● Specimen: Citrated platelet-poor plasma ● When thromboplastin reagent is added to the plasma
○ Citrated plasma (1:9), using 3.2% citrate along with the calcium
○ Recap: citrate poor plasma <10,000/µL ○ Fibrin clot will form
● Preparation:
○ Capped sample should be centrifuged at 1500g
(gravitational force rcf) at room temperature for
10 minutes (CLSI)
■ rcf: Distance of the sample from point
of rotation is regulated
■ This produces a “platelet-poor-
plasma”
● <10,000 plts/uL ● It will transform the sample from a translucent fluid into a
○ PT should be tested within 24 hours gel-like substance
■ On the other hand, aPTT should be ● Right picture: Solid gel like substance in actual
tested in 4 hour
● Shorter time than PT
● Reagents: Reference Value
○ thromboplastin and 0.025 M CaCl2 12 - 14 seconds
■ Source : tissue brain/ tissue extract INR Normal value 0.8 - 1.2
■ Mixture of tissue Factor III, (patients not taking
phospholipids and Ca warfarin)
● In other manufacturers they INR Therapeutic range 2.0 - 3.0
separate Ca in other (patients taking • <2: at risk of thrombosis
containers, some they mix it warfarin) • >3: at risk for bleeding
together in one reagent ● Too much anticoagulant
● Stopwatch
● Water bath Total Volume of Protime Test
● Centrifuge 100 uL Thromboplastin (FActor 3)
● Pipette 100 uL 0.025M CaCl2
100 uL Patient’s Plasma (incubated for 5 mins)
Procedure 300 uL Total
1. Run Normal and Abnormal controls
2. Collect citrated blood. To do this, add exactly 4.5 mL
venous blood 0.5 mL of 3.2% sodium citrate in a graduated Note: Procedure may change depending on the method or machine,
tube, and mix. or reagent used.
● Invert the tube gently for 3-4x. ● Important to read the manual first
● This is done if there is no blue-top ● In some manufacturers they only use 1 reagent which is a
○ If there is, then there is no need to add mixture of thromboplastin (Factor 3) and CaCl
in a graduated tube ○ Generally they require a 200uL of reagent
3. Centrifuge at 1500 rpm for 5 minutes. (tromboplastin + CaCl2)
● Centrifuge at 1500g for 10 minutes (procedure by
CLSI) Prothrombin Time (Diacheck C1) / One Stage PT
4. Separate plasma within 1 hr by aspirating it into a clean
tube and place in a 37°C water bath
● Make sure that all of the solutions are at 37ºC
○ To reflect what is happening inside the
body
5. Into a new and clean test tube (17x75mm), pipette 0.1 mL
thromboplastin and 0.1 mL 0.025 M CaCl2
6. Incubate for at least 10 minutes in a 37°C water bath
7. Pipette 0.1 mL of pre-warmed plasma into the
thromboplastin-calcium mixture in a continuous swift
motion simultaneously starting the stopwatch.
● Once the plasma is pipetted to the reagent, start Materials Needed
the stopwatch immediately ● Specimen: Citrated platelet-poor plasma
8. Leave the mixture in the bath for 5-6 seconds then remove, ● Reagents: thromboplastin reagent
wipe the outside portion immediately. ● Diacheck C1
9. Tilt the tube back and forth against the light until fibrin ○ Detects the formation of fibrin strand
strands can be detected which denote the endpoint then ■ unlike in the manual method wherein
stop the stopwatch. the medical technologist watches out
10. Do the procedure in duplicate. for the formation of fibrin strand
● Do the procedure twice ● Pipette
● To make sure there are no errors ● Centrifuge
● To make sure we can produce the same and
accurate result
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[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
Procedure Note:
1. Collect citrated blood. To do this, add exactly 4.5 mL Because different manufacturers produce different thromboplastin
venous blood 0.5 mL of 3.2% sodium citrate in a graduated reagents
tube, and mix. ● these differences can cause problem when you compare
2. Centrifuge at 1500 rpm for 5 minutes. the PT result from one laboratory to another
● Collect the platelet poor plasma (PRP) ● WHO standardization: oral anticoagulant monitoring
3. Transfer the plasma into a clean tube. ○ Based on anticoagulant expressing PT result in
4. Turn on the instrument and wait until the LED lights up. International Normalized Ratio (INR)
5. Select “PT” as an active test. Calculation:
● Remember that diacheck can check for a variety INR = (𝑷𝑻 𝒑𝒂𝒕𝒊𝒆𝒏𝒕/ 𝑷𝑻 𝒏𝒐𝒓𝒎𝒂𝒍)𝑰𝑺𝑰
of tests (e.g., aPTT, TT) ● PT normal: based on the PT of at least 20 patients with
6. Check calibration. normal value
7. Pre-warm thromboplastin reagent for at least 5 minutes ○ Mean of 20 patients = PT value
● In Diacheck C1 there is an incubator ○ Done by each laboratory
8. Pipette 25µl plasma into a cuvette. INR
9. Press “Optic” ● International Normalized Ratio
10. Pre-warm plasma for 1 minute (press timer). ● Doctors based on INR when patient is under warfarin
● Already in the cuvette ○ Value is standardized
11. Transfer cuvette to measuring area ● INR calculation is intended to yield identical INR result
12. Add 50µl pre-warmed thromboplastin/neoplastine when the same sample is tested into different laboratories
13. Wait for the result. Record. with different manufacturers
○ Calculated using ISI
REPORTING OF RESULTS AND SOURCES OF ERROR ISI
● International Sensitivity Index
● Reflects the sensitivity of the reagent, specific for each
Reporting of Results reagent
● To correct for the variations in thromboplastin reagents, ● Value is given by the manufacturers
○ International normalized ratio (INR) has been ○ No need to calculate, just look for it in the bottle
established and used in reporting of results for of reagent
anticoagulant therapy monitoring. ● Done by a reference plasma provided by WHO to the
● Since PT tests can be sensitive manufacturers
● Prothrombin time results are reported in seconds. ○ Determines sensitivity of reagent
○ However, PT can also be used to monitor ○ Manufacturer makes their own thromboplastin
Coumadin therapy. reagent and determines the ISI by comparing it
■ Expected prolonged clotting time to WHO reference plasma
results ■ Assign reference value to the Lot of
■ Therapeutic range for Coumadin is too the reagent
narrow,
● diligent monitoring of PT is
necessary.

Under Anticoagulation with Overdose Anticoagulation with

Coumadin Coumadin
cause secondary thrombosis cause hemorrhage or bleeding
(rethrombosis)

The desired INR value Patients using mechanical

Figure: Thromboplastin reagent
heart valves.
Reporting Regulated by Clinical and Laboratory Standards
2 to 3 2-5 to 3.5
Institute (CLSI)
Note: If the desired INR value is not reached, then adjustment is
• PT patient and the PT normal: One decimal place
made in the value by the physician.
• ISI Includes: 2 decimal places
•INR should be rounded and reported to: One decimal place
INR is calculated using the formula below: Note:
Minor error in reporting can lead to wrong and erroneous result

Note:
● PT patient = PT of patient in seconds
● PT normal = geometric mean of the PT reference interval
in seconds
● ISI = international sensitivity index, provided by
manufacturers
● PT is reported in seconds

PT/INR Components Results

Component Your Value Standard Range Units Figure: example of Prothrombin Time Activity Results
PROTHROMBIN 13.6 12.2 - 15.0 Sec
TIME In some laboratories, they include in the report the following:
INTERNATIONAL 1.0 0.9 - 1.1 ● control of the day
NORMALIZED RATIO ● patients PT
THERAPEUTIC RANGE: Standard…..2.0 - 3.0 ● PT in activity
HIGH RISK: 2.5-3.5 ● PT ratio
INRs are most often used to monitor warfarin. High risk treatment ● International Sensitivity Index (ISI)
is used in standard treatment failure and in patients with ● International Normalized Ratio (INR)
mechanical valves

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[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
Calculations: ● Congenital single-factor deficiencies of factor X, VII, or V
Prothrombin Time Activity ○ Determination of the defective factor can be done
PTA = (PT control / PT patient) x 100 through mixing studies and special assays
● PT activity is reported in Percentage (%) ● Prothrombin deficiency
Prothrombin Time Ratio ● Fibrinogen deficiency
PTR = (PT patient / PT control)
ACTIVATED PARTIAL THROMBOPLASTIN TIME
Sources of Error
Review: Major components of Hemostasis
Error Solution
1. Overfilled and underfilled Anticoagulant volume must be
evacuated tubes. adjusted if Hct is greater than
55%
2. Overmixing of tubes. Inversions should be gentle and
only for 3 times after blood
collection.
3. Clotted and visibly hemolyzed samples should not be used.
○ Hemolyzed samples can shorten the prothrombin time
4. Plasma lipemia or icterus Alters the optical result of the
can alter the results if specimen
optical instrumentation is
used.
5. Heparin should not be It should be noted on the lab
used request and results if the patient
is receiving heparin therapy.
● Prolonged prothrombin
time
Primary Hemostasis
Factors That Interfere with the Validity of Once a vascular injury occurs, there will be vasoconstriction and the
Clot-Based Test Results collagen is exposed, once collagen is exposed, it will activate the
Specimen Variation Solution platelets and eventually there is formation of a platelet plug.
Blood collection volume less PT and PTT falsely prolonged;
than specified minimum recollect specimen. Secondary Hemostasis
Hematocrit 55% Adjust anticoagulant volume Exposure of blood to the Tissue Factor will activate the coagulation
using formula or nomogram and cascade will lead to the activation of the prothrombin that will be
recollect specimen using new converted to thrombin. Thrombin activates the fibrinogen to form the
anticoagulant volume. fibrin. Together with platelet plug , it will lead to formation of the blood
clot.
Specimen clot All results are affected
unpredictably; recollect
Note:
specimen.
● Blood clot should not stay in the injured area
Visible hemolysis PT and PTT falsely shortened;
● Anti thrombotic control mechanism will regulate the blood
recollect specimen.
clot duration
Icterus or lipemia Measure PT and PTT using a ● Plasmin acts on the blood clot to initiate fibrinolysis and clot
mechanical coagulometer. degradation.
UFH therapy Use reagent known to be
insensitive to UFH or one that
includes a UFH neutralizer such History
as polybrene. ● Describe by Paul Morawitz(1905)
Lupus anticoagulant PT and PTT results invalid; use ○ He identified poor coagulation factors
alternative methods, for instance, ○ Describe the clots as fibrin fibers
chromogenic assays. ○ Fibrin is present in its precursor form, fibrinogen
Incorrect calibration, incorrect Correct analytical error and
dilution of reagents repeat assay.

DIAGNOSTIC IMPORTANCE

Diagnostic Importance of Prothrombin Time

Prolonged PT:
● Vitamin K deficiency
○ Affects Factors II, VII, IX,X
■ Vitamin K dependent factors
○ Malnutrition
○ Use of broad-spectrum antibiotics
■ Normal flora in the GIT produces vit K Note:
● Inhibiting the normal flora FibrinoGEN
can cause the deficiency ● Genesis of the fibrin
○ Malabsorption syndromes ● 1st factor to be discovered
○ Newborns ○ Hence the name Factor I
■ Affected by the diet of the mother FibrIN
■ Don't have enough intestinal bacteria ● Protein of fibers
to produce Vit K
● Liver disease The stimulation of conversion from fibrinogen to fibrin is due to the
○ Factor VII is severely affected because FVIII half- action of Thrombin which has its own precursor form, prothrombin
life is only 6 hours which the 2nd coagulation factor to be discovered
○ Factor VII is found in the extrinsic pathway ● Hence the name Factor II
○ FVII is produced by the liver and due to its half-
life, it cannot compensate for the need of Factor
VII
○ Most of the coagulation factors are synthesized
by the liver
● DIC

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[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
Extrinsic pathway
● Requires something from the outside of the blood (tissue)
● Once there is bleeding and the blood comes in contact
with the tissue to alert the pathway
● Trauma
● Tissue thromboplastin and tissue phospholipids
● Thromboplastin creating the thrombus
● In the blood, factor VII comes in contact with the tissue
factor , Factor VII will be converted to factor VIIa
● Factor VIIa is going to activate the Factor X to become
the Factor Xa that is mediated by calcium
● Factor Xa, Factor V, Phospholipid and Ca2+ will form the
Prothrombinase complex
● Prothrombinase complex activates the prothrombin to
After Factor I and Factor II were discovered, a cascade before the two continue with the cascade until it form the fibrin
factors were also discovered :
● Factor III → Factor XII What to remember:
● But no Factor VI The Extrinsic pathway is …
● Factor XIII is discovered by chance Activated by Tissue factor
○ Important for Fibrin stabilizer , making it insoluble Measured by Protime
Inhibited by Warfarin
Coagulation Cascade
● Each step is stronger than the one before it Note:
○ Strong fibers are formed through the network to ● As the extrinsic pathway is ongoing in action, the intrinsic
stop the bleeding pathway is also working but it takes longer and it is more
○ Secondary hemostasis is important in stopping efficient
the bleeding
○ Platelet activation is pre- requisitein activation Intrinsic pathway
the secondary hemostasis ● Requires a part of the blood vessel
■ Without activation → no secondary ○ Contact factor
hemostasis ■ Known as self endothelial collagen
● Allows the time and steps for regulation ● Contact factor activates the Factor XII to form the Factor
XIIa
Intrinsic Pathway Extrinsic pathway ● Factor XIIa activates the Factor XI to form Factor XIa
● Factor XIa activates the Factor IX to form the Factor IXa
● Intrinsic to the blood ● from outside the blood (i.e. mediated by calcium
● Blood vessel" tissue) ● Factor IXa activates Factor X to convert to Factor Xa
● Depends on the factors ● depends on the factors from mediated by Factor VIIIa, phospholipids and calcium
from within the blood outside the blood ● Eventually it will lead to the formation of fibrin
● more steps "longer ● less steps "shorter
cascade" cascade" What to remember:
● slower, but stronger, more ● faster but less efficient Intrinsic pathway is …
efficient Activated by Self epithelial collagen
Measured by Prothrombin time
Question: Inhibited by Heparin
When you put blood in a test tube, it coagulates using which
pathway? Note:
● a Intrinsic ● Once it reaches to the activation of Factor X, it will be now
● b.Extrinsic known as the common pathway
● c. Both ● Factor VIII is known as the von Willebrand factor which
comes from the platelets
● Factor XIII ( Fibrin stabilizing factor) which converts to fibrin.
Stabilization of fibrin strand is done through cross-linking

Follow up question:
If the blood coagulates in vitro via an intrinsic pathway , How is it
possible if there is no presence of the self endothelial collagen in the
tube?

Fig. Polarized electron micrograph of a platelet clump, it has RBCS (red) , WBC (green),
platelets (white) and Fibrin strands (Orange)

● When factor XII from the blood gets in contact with the
glass, it gets activated

Prothrombin is activated by Prothrombinase complex Clotting factors

Prothrombinase complex: COAGULATION FACTORS
● FactorXA ● Plasma Proteins (B - globulins)
● FactorVA ● Source:
● Phospholipid ○ Liver
● Ca2+ ● Nomenclature: Roman Numerals and the order they were
○ Ionized form discovered
■ Active form of calcium in the body ● according to function, disease, patient that is person
discovered
○ Factor IX is discovered from the patient in 1952,
Stephen Christmas who has this deficiency

Page 8 of 19
[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
Factor Name COAGULATION FACTORS ARE CLASSIFIED INTO:
I Fibrinogen
II Prothrombin FIBRINOGEN GROUP
III Tissue Thromboplastin Not present in the serum
IV Calcium Ions Increased in inflammation
→Acute phase reactants
V Labile Factor
VII Stable Factor These are the following factors :
VIII Antihemophilic Factor ● Fibrinogen (I)
● V
IX Christmas Factor, or Plasma Thromboplastin
● VIII
Component (PTC)
○ Von willebrand factor
X Stuart-Prower Factor ■ Prolongs and sustain the Factor VIII
XI Plasma Thromboplastin Antecedent (PTA) in the serum and acts as the carrier
■ Von willebrand disease
XII Hageman Factor ● Shortened Factor VIII half
XIII Fibrin-Stabilizing Factor (FSF) life
● XIII
○ Found in . platelets
Question:
What is the most heat unstable coagulation factor?
● Factor V PROTHROMBIN GROUP (“1,9,7,2”10,9,7,2”)
○ Labile Factor Vitamin K dependent Factors
○ Disintegrate upon storage All are present in the serum
Vitamin K is in oxidized form
Coagulation factors ● Inactivated form
● Converted to active form by the enzyme Vitamin K
● Fibrinogen group epoxide reductase (VKOR)
● Prothrombin group ○ Targeted by the Warfarin by inhibiting the
● Contact Group enzyme

Note:Vitamin K activates the gamma-glutamyl carboxylase

(GGCX) which is important for the activation of the procoagulants
and anticoagulants.

These are the following factors :

Procoagulants:
● Prothrombin (II)
● VII
● IX.
● X
Note:
● At the bottom, RBCs can be found meanwhile at the top, Anticoagulants
the supernatant is also known as the Plasma ● Protein C
● Without the anticoagulant, the supernatant is known as ● Protein S
the serum and it is separated from the blood clot
CONTACT GROUP
Serum Plasma Activated by the contact with the sub endothelial collagen or the
● It is known as plasma without the ● Plasma can still form negatively charged surface of the glass
clotting factors clot All of them are present in the serum, and formed in the liver
● Cannot clot as the clotting factors ● Has clotting factors
were already used up for the and fibrinogen
formation of the blood clot present Note:
○ Factor II , V, VIII,and ● Requires short time to ● Some books considered High Molecular Weight
fibrinogen prepare Kininogen(HMWK), KALLIKREIN, PLATELET FACTOR
■ Factors mentioned are ● No need for standing 3 (PF3) and GLASS (Negatively. charge surface,
known to the unstable ○ which is a wettable surface) are part of the
during storage contact group
● Requires longer time to prepare
These are the following factors :
○ Needs the coagulation ● XII
cascade to be produced ● XI

Anticoagulation
Question: Prevention of stroke
Why does blood coagulate in vitro via the Intrinsic Pathway? ● Atrial fibrillation
(take note there is no sub-endothelial collagen in the test tube) ○ Rapid or irregular
heartbeat
A. High Molecular Weight Kininogen(HMWK), KALLIKREIN, ● Prosthetic heart valve
PLATELET FACTOR 3 (PF3),GLASS (Negatively. ● IV thrombus
charge surface, which is a wettable surface) ○ Complication of acute
myocardial infarction
Prevention and treatment of ● Deep-vein thrombosis
Note: venous thromboembolism
● Glass should be coated with silicon ● Pulmonary embolism
○ to stop the glass from getting wettable to avoid Embolism
the activation of the coagulation pathway ● Blockage of blood vessels
● Test tubes with anticoagulants are now made up of due to a clot
plastic Treatment of thrombophilic ● Factor V Leiden
○ to avoid the activation of extrinsic coagulation disorder ● Antiphospholipid Ab
pathway
Syndrome
● Platelet membrane is negatively charged
Present prosthetic valve thrombus
○ which supports the assembly of the coagulation
● most common cause why we give anticoagulant to
factors
patients
Page 9 of 19
[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
WARFARIN Most common Vitamin K antagonist in clinical ● The phospholipid mixture, which was historically extracted
practice from rabbit brain, is now produced synthetically.
AKA Coumadin (brand name) ● The activator provides a surface that mediates a
● Oldest anticoagulant conformational change in plasma factor XII that results in
its activation.
● Most common ● Factor XIIa (activated Factor XII) forms a complex with two
● Actions in inhibition of vitamin K epoxide other plasma components:
reductase, recycles Vitamin K after it has ○ high-molecular-weight kininogen (Fitzgerald
been used as coenzyme in coagulation factor) and
pathway: ○ prekallikrein (Fletcher factor).
○ Prothrombin ● These three plasma glycoproteins, termed the contact
○ Factor VII, IX, X, protein C, protein S activation factors
● Challenging to use because of its narrow ○ initiate in vitro clot formation through the intrinsic
therapeutic window pathway but are not part of in vivo coagulation.
● Not predictable with dosage given to the ● The complexed factor XIIa, a serine protease, activates
patient. factor XI (XIa)
○ INR must be check regularly ○ which activates factor IX (IXa).
● Target window for treatment should be within ● Factor IXa forms a complex with factor VIIIa, reagent
INR 2.0-3.0 calcium, and reagent phospholipid.
HEPARIN Antithrombin, inactivation of factor 10a ○ This complex catalyzes factor X.
● The resultant factor Xa forms a second complex with
● To prevent formation of clot ○ calcium, phospholipid, and factor Va
● The molecular structure of heparin which is a ○ catalyzing the conversion of prothrombin to
polysaccharide binds to and activates thrombin.
antithrombin ● Thrombin catalyzes the polymerization of fibrinogen and
● Has extremely high concentration of negative the formation of the fibrin clot
charge which helps its binding to antithrombin ○ which is the endpoint of the PTT.
● The binding induces conformational change ● Most PTT reagents are designed so that the PTT is
○ that enhances the antithrombin in prolonged when the specimen has less than approximately
activating THROMBIN and Factor Xa 30 units/mL of factors VIII, IX, or XI.
Forms of Heparin
● UNFRACTIONATED HEPARIN
○ Made up of 45 saccharide units
○ Acts on Thrombin
○ UFH depends on dose-response
relationship so it has to be monitored
closely by aPTT
● LOW-MOLECULAR WEIGHT HEPARIN
○ 15 saccharide units
○ Acts on Factor Xa

OVERVIEW of COAGULATION TESTS

1. Clotting time
2. Prothrombin time (PT) Figure: Partial Thromboplastin Time Assay
● Evaluates extrinsic and common pathways
3. Activated Partial Thromboplastin Time (aPTT)
● Evaluates intrinsic and common pathways
4. Stypven Time/ Russell’s Viper Venom Time
5. Thrombin Time
6. Reptilase Time Test
7. Duckert’s Test
8. Mixing study
9. Fibrinogen level
10. Factor assays
11. 5.o M urea clot solubility

Why do we need to do COAGULATION TESTS?

1. Assess Patient with unexplained bleeding
2. Assess Patient ready for surgery (preoperative testing)
● Avoid risk of bleeding after surgery
3. Patient taking anticoagulants
● Monitoring anticoagulant
● Too much anticoagulation
○ Lead to bleeding ● aPTT measures intrinsic and common pathway
● Not enough coagulation ○ Intrinsic: FXII, FXI, FIX, FVIII
○ Lead to thrombosis ○ Common: FX, FV, FII, FI

Principle of aPTT Test

● Measures the function of the other contact activation
pathway or intrinsic pathway, as well as common pathway
● The activated Partial Thromboplastin Time is employed to:
○ monitor the effects of Unfractionated Heparin
○ to detect Lupus Anticoagulant and specific
coagulation factor antibodies
■ such as antifactory VIII antibody.
● The PTT is also prolonged in all congenital and acquired
procoagulant deficiencies
○ except for deficiencies of factor VII or XIII.
● The PTT reagent contains Partial Thromboplastin
(phospholipid without tissue factors) + Calcium and a
negatively charged particulate activator such as: ● Upon the addition of Ca and partial thromboplastin
○ silica, kaolin, ellagic acid, diatomaceous earth or (phospholipid without tissue factor) and activating agent in
celite in suspension. the plasma, it will turn into a fibrin clot
Page 10 of 19
[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
aPTT ● Mix gently by shaking the tube and incubate in
Reagent Calcium + Partial Thromboplastin the water bath for exactly 3 minutes.
(phospholipids without the tissue factors) ○ Must time it accurately
Activating agents silica, kaolin, ellagic acid, diatomaceous ○ This will not produce a clot yet
earth or celite 7. Add 0.1mL pre-warmed 0.025M calcium chloride using a
No standardization like the PT pipette simultaneously starting a stopwatch.
● Gently mix the tube
● Without tissue factor, as aPTT does not measure extrinsic ● Addition of CaCl leads to the formation of clot
pathway but intrinsic 8. After 30 seconds, remove from the water bath and gently
● Kaolin: most common surface activator or activating agent tilt back and forth until final gel formation then stop the
used stopwatch.
● Diatomaceous earth: sand, purified to become an activator 9. Repeat the test using another aliquot of the test plasma and
report the average of the tests.
10. Run 2 control tests on standard plasma.
aPPT METHODS AND PROCEDURES ● If aPTT is prolonged, do two tests on 1:1 mixture
of normal plasma and patient’s plasma.
Sample Requirements
● The anticoagulant of choice when testing for aPTT is Total Volume of aPTT
trisodium citrate. Citrated plasma: 100 µL 0.025M CaCl2
○ Preserves the labile clotting factors V and VIII 100 µL Patient’s Plasma
better 100 µL Partial thromboplastin reagent (phospholipid & activator)
○ The most satisfactory for platelet aggregation
studies 300 µL Total
○ More sensitive to the effects of heparin and
therefore, preferred for tests to monitor heparin Semi-automated Method
therapy ● Convenient than manual method
● Standard ratio of anticoagulant to blood ● The device will do the incubation, timing and detection of
○ 9:1 blood to anticoagulant clot
● After centrifugation, separate the upper ¾ layer of plasma ○ All you have to do is to dispense the sample and
from the sample the reagent, then wait for the machine to give out
● Refrigerated centrifuge is advised (2-4˚C) the report.
● Samples that can’t be tested within 2 hours should be ○ There is a need for calculations
frozen rapidly at -20˚C or lower ● Activated Partial Thromboplastin Time using BIOBAS10
Machine
Test Reagent
The PTT reagent contains: 1. Withdraw blood in citrated vacutainer
phospholipid the phospholipid mixture, which 2. Centrifuge at 2000 rpm for 10 minutes
(previously called partial was historically extracted from 3. Turn on the instrument
thromboplastin or cephalin) rabbit brain, is now produced 4. Select “aPTT” as an active test by pressing < or > keys.
synthetically. Then press ENTER.
activator the activator provides a surface ● This device does not only test for aPTT, also
(negatively charged particulate that mediates a conformational measures the Protime, thrombin time, and
such as silica, kaolin, ellagic change in plasma factor XII that fibrinogen
acid, diatomaceous earth or results in its activation. 5. Prepare the reagents R1 and R2
celite in suspension) ● R1: Partial thromboplastin
● R2: 0.025M CaCl
Test Procedure
How are they prepared?
● To initiate contact activation, 50 or 100 mL of warmed (37° ● The manufacturer will send vials containing lyophilized
C) reagent consisting of phospholipid and particulate form of regents (dehydrated regent)
activator is mixed with an equal volume of warmed platelet- ○ You can prepare by adding distilled water
poor plasma. ○ The volume is according to the manufacturer’s
● The mixture is allowed to incubate for the exact instruction
manufacturer-specified time, usually 3 minutes. ○ Sit for 30 minutes before using the reagent
● Next, 50 or 100 mL of warmed 0.025 M calcium chloride is
forcibly added to the mixture, and a timer is started.
6. Dispense 100μl plasma into cuvette
● When a fibrin clot forms, the timer stops, and the interval is
7. Add 100μl of R1 in the cuvette
recorded.
● Inside a cuvette has metal rod
● Timing may be done with an automatic electromechanical
○ Prior in performing the procedures,
or photo-optical device.
make sure that the metal rod is present
● Results are reported to the nearest tenth of a second, along
inside the cuvette
with the PTT reference interval.
○ Metal rod is the one who will detect the
clot
SAMPLE OF MANUAL AND SEMI-AUTOMATED METHOD 8. Place cuvette to the measuring channel to activate
chronometer and close cover for proper incubation. Press
Manual Method Start. The machine will time for incubation which is 5
minutes.
● In manual method, you have to do everything yourself:
● Make sure to close the cover
timing, incubation, check for the clot
○ If open, the temperature will fluctuate
9. After the incubation, the machine will flash GO-S. This is
1. Obtain 5mL venous blood
your signal to add 100μl of R2 (0.025M CaCl). Hastily
● Syringe method
dispense R2. The machine will start timing right after the
2. Add 4.5mL to 0.5mL 3.2% sodium citrate solution in a clean
addition of R2.
tube and mix gently by inversion.
10. The chronometer stops when a clot is detected. After
● Collect Citrated blood.
analysis, time, percentage activity and INR will be displayed
3. Centrifuge at 1500g for 10 minutes or 2000 rpm for 10
on the screen. Wait for the result and record APTT
minutes
accurately.
● Different units for different speed
4. Transfer plasma to another clean tubes within 1 hour and How will the machine detect the clot?
test within 2 hours ● The machine has magnetic field to shake the metal rod
5. Pre-warm aliquots of unknown and standard plasma ● As we add CaCl and will start to form clot, the metal rod
● At 37°C water bath will stop moving
6. Into a clean test tube containing 0.1mL pre-warmed test
plasma, 0.1mL pre-warmed reconstituted partial 11. Identifies and segregates waste generated and dispose
thromboplastin. them properly.
Page 11 of 19
[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
Fully Automated Machine Sources of Error

1. Sample Collection and Preparation

● Traumatic venipuncture can cause premature activation of
the clotting process before testing of the sample.
● Contamination of the specimen with tissue thromboplastin,
which is a potent clot activating substance found in fluids
that escape from tissue spaces will activate the extrinsic
pathway of clotting and causes erroneous test results.
● More convenient because it uses barcode reader
Anticoagulant volume must be adjusted if:
● The samples has barcodes and the machine will
Hematocrit >0.55 High Hct: Results to falsely prolonged
automatically read if what test needs to be performed
L/L or 0.2 L/L ● There is less plasma volume → less
● Used for aPTT, PT, Fibrinogen, and Thrombin Time
● You only need is to centrifuge the sample, prepare the Poor coagulation factors present
Platelet Plasma, and place the sample inside Low Hct: Results to falsely shortened
○ The machine will do the dispensing ● More plasma volume → more
○ Inside the machine contains the reagents, times coagulation factors present
the test, and do the calculation Hemolysis Hemolyzed RBCs act like tissue
(shortened) thromboplastin. This can be due to:
aPTT using Diacheck Machine ● Excessive stasis through prolonged
1. Withdraw blood in citrated vacutainer tourniquet application
2. Centrifuge at 1000 rpm for 5 minutes ● Moisture or contamination in the
3. Turn on the instrument and wait for the LED light needle, syringe, or blood container
4. Select “PTT” as active test ● Using needles with too small a bore
5. Pre-warm Calcium chloride for at least 5 minutes ● Frothing of sample due to entry of air
6. Pipette 25μl plasma into cuvette
7. Add 25μl aPTT reagent plasma ● Expelling of blood from syringe
8. Incubate for 5 minutes through the needle
9. Transfer cuvette to measuring area ● Excessive and vigorous mixing of
10. Press “Optic” blood with the anticoagulant
11. Add 20μl pre-warmed calcium chloride and simultaneously Unexpected Heparin
start timing Contamination
12. Wait for the result and record aPTT accurately (Lengthen)
13. Repeat the procedure and report the average of the tests
14. Identifies and segregates wastes generated and dispose 2. Reagent Preparation
them properly ● We prepare our own reagent since manufacturer’s provides
lyophilized form reagents
STANDARD REFERENCE VALUE AND SOURCES OF ● Improper storage, water impurities, incorrection dilution
ERROR
3. Instrumentation
Standard Reference Values ● Flailing light source, fluctuations in temperature, loss of
● The aPTT Reference Interval is 25–35 seconds (Keohane calibration of tubing, contamination
et al, 2020). ● When using semi-automated, and fully automated
○ Others: <45 sec is acceptable
● This is typical, but each center must establish its own DIAGNOSTIC IMPORTANCE
interval for each new lot of reagent, or at least once a year.
○ This may be done by testing a sample of 30 or The physician orders 1. hemorrhagic disorder
more specimens from healthy donors of both a PTT when 2. recurrent thrombosis
sexes spanning the adult age range over several investigating: 3. presence of an autoimmune disorder
days and computing the 95% confidence interval (possibility of Lupus Anticoagulant)
of the results. The PTT result is 1. prothrombin; factors V, VIII, IX, X, XI,
● The PTT reference interval varies from site to site. prolonged when there or XII; or
is a deficiency of one 2. fibrinogen when the fibrinogen level
Factors Affecting the reference values or more of the is 100 mg/dL or less
1. Patient population following coagulation
2. Type of reagent factors:
3. Type of instrument Factor VII and factor XIII deficiencies have no effect on the PTT. Deficiencies of
4. pH and purity of the diluent factor XII, prekallikrein, or high-molecular-weight kininogen prolong the PTT but
do not associate with bleeding.

Example of a Result
Pathology
1. Deficiencies of factors VIII, IX, or XI;
• Hemophilia A
• Hemophilia B
• Rosenthal syndrome
• The deficiencies in these coagulation factors that
belongs to intrinsic pathway → Prolonged aPTT
time
2. Presence of a specific inhibitor
● Only concludes control and patient’s aPTT ● Anti-factor VIII or anti-factor IX;
● Specific inhibitor: antibody against a factor
3. Nonspecific inhibitor, such as LAC (lupus anticoagulant),
● Prolonged aPTT time
● LAC is anti-phospholipid
○ Neutralize a part of aPTT reagent
(phospholipid) → Prolonged
4. Immunoglobulin with affinity for phospholipid-bound
proteins
5. Interfering substances, such as fibrin degradation products
(FDPs) or paraproteins (present in myeloma)
● They can inhibit phospholipid
Page 12 of 19
[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
6. DIC: consumption of multiple procoagulants. Lab Findings
● PTT results must be confirmed using:
○ the D-dimer, platelet count, and
INR Normal
erythrocyte morphology
APTT Prolonged
● DIC decreased platelet count
● DIC is also in a clinical significance in PTT Corrected by Mixing Studies
○ Therefore, in DIC, both PT and aPTT ● Used to determine which factor
→ prolonged deficiency the patient has
7. Vitamin K deficiency diminishes activities of procoagulant
factors II (prothrombin), VII, IX, and X, and the PTT is Since Factor VIII and IX has defects, APPT are affected
eventually prolonged. ● APPT is measured by extrinsic pathway
● FII, FX, FIX: Common pathway → involved in
PTT 3. Rosenthal Syndrome (Hemophilia C)
● Inherited hemorrhagic condition that is caused by
1. Disseminated Intravascular Coagulation (DIC) coagulation Factor XI deficiency
● Mild form of hemophilia which is predominant in Ashkenazi
Jews
● Occurs in 1/100,000

Lab Findings

Protime Normal
Thrombin Time Normal
APTT Prolonged

4. Vitamin K Deficiency
● Vitamin K stands for “koagulaitions vitamin”, German for
1. Cause of DIC: Systemic exposure to procoagulant factor
clotting vitamin
2. If it is triggered, it will activate the coagulation cascade →
● Fat soluble, requires intact bowel and pancreatic function
widespread of microthrombi → (a) consumes the
for effective absorption
coagulation factors and platelets
3. Decrease coagulation factor and platelets → bleeding
Source Green vegetables like spinach, kale, brussel
● We can’t stop bleeding if there is no coagulation
sprouts
factors since they are consumed
4. Widespread of microthrombi → (b) widespread fibrinolysis Cause Malnutrition, antibiotics use which kills of
● The presence of clot formation regulate the clot beneficial gut bacteria, malabsorption
by our body → Triggers the fibrinolysis
5. (a) Consumption of coagulation factors and platelets, and Lab Findings
(b) widespread of fibrinolysis will end up to organ damage
● Prolonged aPTT PT Prolonged
● We need Vit. K for extrinsic pathway
Lab Findings APTT Normal or prolonged
Mixing Study Normalize
Platelets Decrease ● We can confirm for Vit. K deficiency
INR, APTT Prolonged
● Factor deficiency due to consumption 5. Acquired Factor Inhibitor
Fibrinogen Decreased ● Rare condition which is clinically similar to hemophilia,
● Fibrinogen, together with coagulation factors ○ but is caused by antibodies that either inhibit or
and platelets, will be used up increase the clearness of a clotting factor (usually
D-dimer Increased factor VIII)
● Fibrin degradation product ● Associated with malignancy, autoimmune disorders
● Widespread of fibrinolysis → fibrin ● Diagnosis is based on history
degradation product → D-dimer
Lab Findings
2. Hemophilia
● Bleeding caused by a deficiency in a clotting factor PT Variable
● Depends what factor are targeted by
antibodies
● Ex: Factor VIII are targeted → affected are
extrinsic pathway → prolonged
APTT Variable
● Depends on which factor is targeted
● Depends what factor are targeted by
antibodies
Mixing Study Do not normalize

6. Advanced Liver Disease

Common Forms ● Decrease in both procoagulants and anticoagulant factor,
dysregulated hemostasis
Hemophilia A Deficiency in Factor VIII ● INR do not accurately reflect risk of bleeding in cirrhosis
Occurs in 1/4000 ● Coagulant factors are produced by the liver → impaired
Hemophilia B Deficiency in Factor IX function of liver → defective production of coagulant factors
Occurs in 1/30,000
Both are X-linked recessive Lab Findings
● Only males manifest this disorder
● Females can only be a carrier PT Prolonged
APTT Normal or Prolonged
Diagnosis Mixing Study Normalized
● Consistent bleeding history, family history

Page 13 of 19
[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
7. Anticoagulant Medications
● Heparin, LMWH (Low molecular weight heparin), warfarin

Lab Findings

PT Variable
APTT Variable
● Depends on specific medicine and dose
● Ex: Insufficient dose → may shortened PT
and APTT
○ Overdosed → may prolonged PT and
APTT
Mixing Study Normalize

8. Lupus Anticoagulants
● Decrease in both procoagulants and anticoagulant factors, Clinical Significance: Prolonged TCT
dysregulated hemostasis 1. Hypofibrinogenemia (also in DIC)
● Described by Feinstein and Rapaport in 1972 ● Fibrinogen is decreased in level
● Antiphospholipid Syndrome and Systemic Lupus ● Happens in Disseminated intravascular
Erythematosus coagulopathy
● Due to autoantibodies that inhibit phospholipid-dependent ○ Fibrinogen is continuously and
coagulation in vitro excessively consumed
● Directed against phospholipid-binding protein (β2-GPI) ● Prolonged thrombin time
2. Afibrinogenemia
Screening Test aPTT ● Congenital absence of the fibrinogen production
Confirmatory Test platelet neutralization procedure ● Prolonged thrombin time
(patient's plasma + PL excess) 3. Dysfibrinogenemia (Liver disease,neonates)
Serological Test We can test for Phospholipid antibodies ● Can produce fibrinogen but dysfunctional
● Thrombin time measures for functional fibrinogen
→ if dysfunctional → Prolonged thrombin time
APTT v.s. PT
4. Hypoalbuminemia
APTT PT
● Happens in kidney disease
Measures Intrinsic and common Extrinsic and common ● Decrease in fibrinogen concentration →
pathway pathway Prolonged thrombin time
Intrinsic/ Intrinsic: occurs in Extrinsic: occurs in 5. Raised concentrations of FDP as encountered in DIC or
extrinsic vivo and in vitro vivo only liver disease
Coagulation APTT (4 letters) PT (2 letters) ● Fibrinogen concentration is decreased
factors ● Factors 12, ● Factors 3 & 7 ○ Clotting process is continuously
11, 9 & 8 consumed
Normal < 45 seconds < 15 seconds ● Prolonged thrombin time
range 6. Presence of antithrombotic materials such as FDPs,
(varies in the paraproteins, or UFH
laboratory ● The principle in thrombin time is the conversion
and reagent of fibrinogen to fibrin clot with the aid of thrombin
used) ● If antithrombin → prolonged thrombin time
Requires Surface activator and Tissue extract & Ca 7. Presence of the oral direct thrombin inhibitor - Dabigatran
Ca ● Prolonged thrombin time
Prolonged in Heparin therapy Vitamin K deficiency ○ The effect of Dabigatran is against
Hemophilia Vitamin K antagonist thrombin (Direct inhibitor)
Liver disease (warfarin) 8. Pregnancy and newborns
Antiphospholipid Liver disease
antibody syndrome Factor deficiency Pregnancy
Warfarin Antiphospholipid ● In pregnancy, for babies to “kapit” to the uterus, we would
DIC antibody syndrome be needing an increased concentration of fibrinogen
Heparin ● That is why pregnant women have a decrease in fibrinogen
DIC levels especially during labor.
● So the thrombin time is expected to be prolonged.
THROMBIN CLOTTING TIME
● Measures the availability of functional fibrinogen Newborns
● Principle: Conversion of fibrinogen to fibrin ● Their liver is still not fully functional
● Qualitative and quantitative measurement for fibrinogen ○ Take note: fibrinogen is produced in the liver.
○ Since it measures the concentration and function ● Low function of liver = low fibrinogen level
● A measured amount of thrombin is added to plasma to ● Thus, thrombin time will be prolonged
directly activate fibrinogen
● The length of time for a fibrin clot to form is recorded as the 9. Multiple myeloma
thrombin time
● The endpoint is the formation of the clot, it is only specific Multiple Myeloma Products: Light chain or paraprotein
for fibrinogen ● Could inhibit fibrinogen function
○ Belongs to coagulation factor assays ● So the light chain (kappa and lambda) will attach to the
● 100 μL citrated PPP fibrinogen molecule, therefore inhibiting its function
● Reagent: 200 μL thrombin-CaCl2 ● Fibrinogen will be dysfunctional resulting in prolonged
○ Thrombin will convert fibrinogen to fibrin clot thrombin time.

Reference Value
15-20 seconds

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[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
MIXING AND SUBSTITUTION STUDIES Aged Serum
● Absent: Fibrinogen groups
➔ Factors I, V, VIII, and XIII
Preparation of Blood Derivatives ○ It is still serum so we expect that the fibrinogen
● Used for Substitution and Mixing Studies group will be absent
● Absent: Prothrombin Coagulation Factor
Patient's Centrifuged citrated whole blood at 1500 rpm for ➔ Factor II
plasma 5 mins. Mix 0.1 mL plasma and 0.9 mL saline (1:10 ■ Since it is aged serum, not fresh
ratio). anymore
Adsorbed Incubate 1 mL plasma w/ 50 mg barium sulfate ■ Prothrombin coag factor (Factor II) is
plasma at 37°C for 15 mins with frequent mixing. consumed
Centrifuge at 2,000 rpm & use immediately.
● Some of the coag factors can be absorbed Absent in…
using barium sulfate. Adsorbed plasma Aged serum
● Remember the Prothrombin group: Prothrombin Group Fibrinogen Group
○ Factors II, VII, IX, and X are Vitamin K ● FII, FVII, FIX, FX ● FI, FV, FVIII, FXIII
dependent and at the same time they Prothrombin Group (absent in both)
are absorbed using barium sulfate.
Aged Incubate plasma at 37°C for 2-3 days & may be Mixing Studies
normal stored in aliquots at 35°C. Prothrombin time (Circulating Anticoagulant Screen, Screening for
plasma must be over 60 seconds. Circulating Inhibitor)
● 2-3 days: that is why it is called aged
● Aliquots: can be separated in different vials A mixing study is performed to measure aPTT in the patient’s plasma
● Prothrombin time over 60 seconds: ● Mixing an equal volume of the patient’s plasma with the
prolonged because it was stored as aged normal pooled plasma
normal plasma. ● Repeating the APTT test immediately and after 37°C of
○ Aged plasma caused the coag factors to incubation
disintegrate or damage. So we expect ● Determine presence of circulating anticoagulants or
that the prothrombin related factors are circulating inhibitor
decreased in concentration.
Aged Incubate clotted whole blood at 37°C for 3 hrs. Further mixing study could be performed with substitution
serum Add 1 vol 0.1M sodium citrate to 9 vol of blood. studies:
Incubate at 37°C for 2 hours. Centrifuge for 10 ● To identify the specific factors that are deficient
mins and use immediately. ● Performed when both PT & aPTT are prolonged
● We were able to obtain serum from incubated ● To differentiate a factor deficiency from factor inhibitors,
whole blood that clotted. such as lupus anticoagulant, or specific factor inhibitors
(dependent on the coag factor targeted), such as antibodies
directed against factor VIlI
Summary of Factors Found in Blood Derivatives
How do you prepare fresh plasma, aged plasma, adsorbed plasma
and aged serum?
● Proceed to Preparation of Blood Derivatives table

Indicators
1. Correction (shortened than reference range)
● Result of prolonged aPTT:
○ Factor deficiency
■ Hereditary
■ Acquired
● Warfarin therapy
(oral
anticoagulant)
P: Present; A: Absent ● Liver disease
Fresh Plasma ● Vitamin K
● All coagulation factors are present deficiency
○ Especially when you obtain the citrated whole 2. Partial or no correction
blood and centrifuge the sample ● Circulating inhibitor
Aged Plasma ○ heparin, lupus inhibitor, VIll & IX
● Absent: Factor V and VIII inhibitor
○ Labile Factors ■ Anti Factor VIII is the most
○ Will be absent over time common inhibitor
Adsorbed Plasma
● Absent: Prothrombin group or Vitamin K dependent group Correction After mixing study, there will be shortened
➔ Factors II, VII, IX, and X time than the reference range
○ Adsorbed Plasma using barium sulfate (BaSO4) Partial or no Above reference range result
will be absorbing the prothrombin group or correction
vitamin K dependent groups or prothrombin
group Substitution Studies
○ BaSO4 disintegrates or deteriorates the Vitamin ● Used to identify specific coagulation factor deficiencies
K dependent factors ○ Using the table of factors found in blood
Fresh Serum derivatives
● Absent: Fibrinogen groups ● May be performed using adsorbed plasma and aged serum
➔ Factors I, V, VIII, and XIII with the aPTT to identify deficiencies of blood coagulation
○ Since we are obtaining a serum sample, there ● May also be performed using adsorbed plasma with the PT
will be clot formation to identify a factor VIl deficiency
○ During clot formation, fibrinogen groups are
being consumed.
● <20% remaining concentration: Prothrombin Coagulation
Factor
➔ Factor II
○ Prothrombin as a coagulation factor is partially
consumed in the clotting process.
Page 15 of 19
[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
Exercises: Sample Mixing and Substitution Studies ● Why not Factor II?
Note: ○ Factor II or Prothrombin is not detected by
● 1st step is to predetermine what factor is deficient with PT, Thrombin Time
APTT, and TT ○ If thrombin time is involved then FII can be
● 2nd step is to confirm with mixing studies (with the help of included
factors found in blood derivatives table) ● Why not Factor V?
○ By looking on what facotrs involved in the ○ It is also not detected by Thrombin Time
plasma or serum used for sample ● Why not Factor VIII?
○ It is also not detected by Thrombin Time
Case 1. Determine the Factor deficiency. ○ Also since the problem is in the common
● PT- Abnormal pathway while Factor VIII belongs to the
● APTT-Normal intrinsic pathway
● TT-Normal ● Why not Factor XIII?
○ It is also not detected by Thrombin Time
● Normal Plasma- Corrected
● Adsorbed Plasma- Not corrected
● Aged Serum- Corrected Case 3. Determine the Factor deficiency.
● PT- Normal
Note: focus on not corrected and what are the coagulation factors ● APTT-Abnormal
affected ● TT-Normal

Case 1. Solution ● Normal Plasma- Corrected

● Pathway affected: Extrinsic ● Adsorbed Plasma- Not corrected
● PT- Abnormal ● Aged Serum- Corrected
○ PT is specific for Factor VII deficiency
identification Case 3. Solution
● APTT-Normal ● PT- Normal
○ No problem with intrinsic pathway ● APTT-Abnormal
● TT-Normal ○ Problem with intrinsic pathway
○ No problem with fibrinogen ■ Factors VIII, IX, XI and XII
● TT-Normal
Prediction: Factor VII, confirm with results of patient’s plasma mixed
with the following samples: ● Normal Plasma- Corrected
(Tip: Check which sample is not corrected and write the ● Adsorbed Plasma- Not corrected
corresponding coag factors which are absent) ○ Factors II, VII, IX, and X
● Aged Serum- Corrected
● Normal Plasma- Corrected
● Adsorbed Plasma- Not corrected Final Answer: Factor IX Deficiency
○ Absent: Factors II, VII, IX, and X (Prothrombin ● Why not Factor II?
group) ○ Common pathway
● Aged Serum- Corrected ● Why not Factor VII?
○ Extrinsic pathway
Confirmed and Final Answer: Factor VII Deficiency ● Why not Factor X?
● Why not Factor II? ○ Common pathway
○ Common pathway is normal
■ Normal APTT
● Why not Factor IX?
○ Intrinsic pathway is normal
■ Normal APTT
● Why not Factor X?
○ Same for Factor II, common pathway is normal
■ Normal APTT

Case 2. Determine the Factor deficiency.

● PT- Abnormal
● APTT-Abnormal
● TT-Abnormal

● Normal Plasma- Corrected

● Adsorbed Plasma- Corrected
● Aged Serum- Not corrected

Case 2. Solution
● Pathway affected: Common pathway
● PT and APTT- Abnormal Annotater’s note: this figure and the info below were copy pasted from the internet to aid
○ Problem with common pathway with the exercises.
Figure source:
○ Common pathway involves: ● Coagulation Cascade - Stepwards. (2016). Stepwards.
■ Factors X, V, II, and I https://www.stepwards.com/?page_id=866
● https://www.pinterest.com/pin/147141112798352648/
● TT-Abnormal
○ This measures for fibrinogen so this means
there is a problem with fibrinogen Intrinsic Pathway aPTT Factors XII, XI, IX, and VIII.
■ Factor I (plus Pre-K and HMWK)
Extrinsic Pathway PT Factor VII
Prediction: Factor I (or fibrinogen)
Common Pathway PT and Factors X, V, II, and I
● Normal Plasma- Corrected aPTT
● Adsorbed Plasma- Corrected
● Aged Serum- Not corrected
○ Absence of fibrinogen group and Prothrombin
■ Factors I, II, V, VIII, and XIII

Confirmed and Final Answer: Factor I Deficiency (Fibrinogen

Deficiency)
Page 16 of 19
[MLS12b] Module 5 CLOT-BASED SCREENING TESTS
VENOM ACTIVATED ASSAYS Russell’s Viper Venom Time
● Dilute Russell Viper Venom Test / Stypven Time
Reptilase Time ● Used to detect deficiencies in the common pathway
○ Prothrombin, fibrinogen, FV, and FX are detected
● Reptilase
here
○ thrombin-like enzyme found in the venom of the
○ Especially FX and FV
Bothrops atrox snake
○ Capable of converting fibrinogen to fibrin
○ It is unaffected by therapeutic heparin Reagents
■ This means that this procedure is ● Reagent: Daboia russelii East Indian viper venom (0.1mL)
helpful in testing for functional
fibrinogen when thrombin time is ● 25 mM CaCl
prolonged because of the presence of ○ Surface negative charge and activator
heparin and other antithrombins. ● 0.1mL Phospholipid
● Measures functional fibrinogen ○ Surface

Principles Note: We need a complex formation so we will be needing a surface

● When reptilase is added to plasma, it acts by releasing FP ● by adding phospholipid and CaCl
A (fibrinopeptide A) from the fibrinogen.
○ Recap: FP A will be cleaved to eventually form Sample
the fibrin monomer, converting it to fibrin ● 0.1mL Plasma
polymer, resulting in formation of the fibrin clot. ○ PPP (platelet poor plasma)
● The monomers polymerize end-to-end, forming a clot.
Note:When the reagent is added to the plasma containing lupus
Reagents anticoagulant, some of the phospholipid (reagent) in the test will be
● 0.1mL Atroxin (buffered Bothrops atrox venom) + 0.2mL neutralized by the lupus anticoagulant.
PPP (platelet poor plasma) ● Thus, limiting the amount of phospholipid available in the
○ And then, wait for the endpoint or formation of the reagent for the coagulation
clot ● Prolonged Stypven Time.

Reference Range Lupus inhibitor or anticoagulant → anti-phospholipid

18-20 seconds

Thrombin Time Reptilase Time Principle

FSPs, FSPs, Prolonged Prolonged ● Detect deficiency in the coagulation factors in the common
Paraprotein pathway
Hypofibrinogenemia Prolonged Prolonged ● Russell viper venom in the presence of FV, phospholipid,
Dysfibrinogenemia Prolonged Markedly and calcium will activate FX and begin the coagulation
prolonged mechanism at the point of conversion of prothrombin to
Immunologic anti- Prolonged Normal thrombin
thrombins ○ Thrombin will eventually activate fibrinogen to
Heparin therapy Prolonged Normal form the fibrin clot.
Use For patients
undergoing heparin Reference Range
therapy or 30-35 seconds
antithrombin ● Prolonged: neutralization of venom by lupus inhibitor
medications ○ Presence of lupus anticoagulant in the patient’s
plasma
Thrombin Reptilase II VI IX X
Cleaves FP A and FP B FP A only

Reptilase Time
● Insensitive to UFH (unfractionated heparin) and FXIll
deficiency
● Occasionally employed to rule out UFH in a patient who has
a prolonged PTT
● Detect hypofibrinogenemia or dysfibrinogenemia in patients
receiving UFH
● May be used to work up a bleeding patient whose factors
VIll, IX, and Xl are normal

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[MLS12b] Module 5 CLOT-BASED SCREENING TESTS

SUMMARY TABLE
PT vs APTT PT APTT TT
Test for which Pathway of Extrinsic, common Intrinsic, common fibrinogen
Coagulation?
Factors Affected? Common pathway: X, V, Prothrombin (II), Fibrinogen (I)
VII XII, XI, IX, VIII (Pre-K, HMWK)
Used to monitor which anticoag Warfarin, coumadin, coumarin Heparin
therapy?
Principle of the test When tissue extract or thromboplastin (serve Measures the function of the other contact
as the TF) is added to platelet-poor plasma activation pathway or intrinsic pathway, and
among with calcium, it reacts with Factor VIIa common pathway
to convert X to Xa along with Va, phospholipid
and calcium that converts prothrombin to Partial thromboplastin + activators is added
thrombin to platelet-poor plasma along with calcium it
reacts with Factor XII → Factor XIIa forms a
complex Factor XIIa (activated Factor XII)
forms a complex with two other plasma
components HMWK and prekallikrein → The
complexed factor XIIa, a serine protease,
activates factor XI (XIa) → Factor IXa forms a
complex with factor VIIIa, reagent calcium,
and reagent phospholipid → The resultant
factor Xa forms a second complex with
calcium, phospholipid, and factor Va →
common pathway → converts prothrombin to
thrombin
Specimen Manual Method: Citrated Platelet-Poor Plasma Manual Method: Venous blood + sodium
Diacheck: Citrated Platelet-Poor Plasma citrate solution
Diacheck: Citrated Platelet-Poor Plasma
Reagent Thromboplastin Partial thromboplastin, activators (silica, 200 uL
kaolin, ellagic acid, celite) thrombin-
CaCl2
Manual Procedure Reagent: Thromboplastin and 0.025M CaCl2 1. Obtain 5mL venous blood
1. Run Normal and Abnormal controls ● Syringe method
2. Collect citrated blood. To do this, add 2. Add 4.5mL to 0.5mL 3.2% sodium
exactly 4.5 mL venous blood 0.5 mL of citrate solution in a clean tube and mix
3.2% sodium citrate in a graduated tube, gently by inversion.
and mix. ● Collect Citrated blood.
3. Centrifuge at 1500 rpm for 5 minutes. 3. Centrifuge at 1500g for 10 minutes or
4. Separate plasma within 1 hr by aspirating it 2000 rpm for 10 minutes
into a clean tube and place in a 37°C water 4. Transfer plasma to another clean tubes
bath within 1 hour and test within 2 hours
5. Into a new and clean test tube (17x75mm), 5. Pre-warm aliquots of unknown and
pipette 0.1 mL thromboplastin and 0.1 mL standard plasma
0.025 M CaCl2 6. Into a clean test tube containing 0.1mL
6. Incubate for at least 10 minutes in a 37°C pre-warmed test plasma, 0.1mL pre-
water bath warmed reconstituted partial
7. Pipette 0.1 mL of pre-warmed plasma into thromboplastin.
the thromboplastin-calcium mixture in a ● Mix gently by shaking the
continuous swift motion simultaneously tube and incubate in the
starting the stopwatch. water bath for exactly 3
8. Leave the mixture in the bath for 5-6 minutes.
seconds then remove, wipe the outside 7. Add 0.1mL pre-warmed 0.025M calcium
portion immediately. chloride using a pipette simultaneously
9. Tilt the tube back and forth against the light starting a stopwatch.
until fibrin strands can be detected which 8. After 30 seconds, remove from the
denote the endpoint then stop the water bath and gently tilt back and forth
stopwatch. until final gel formation then stop the
10. Do the procedure in duplicate. stopwatch.
9. Repeat the test using another aliquot of
the test plasma and report the average
of the tests.
10. Run 2 control tests on standard plasma.
● If aPTT is prolonged, do two
tests on 1:1 mixture of
normal plasma and patient’s
plasma.
Diacheck Reagent: Thromboplastin Reagent 1. Withdraw blood in citrated vacutainer
2. Centrifuge at 1000 rpm for 5 minutes
1. Collect citrated blood. To do this, add 3. Turn on the instrument and wait for the
exactly 4.5 mL venous blood 0.5 mL of LED light
3.2% sodium citrate in a graduated tube, 4. Select “PTT” as active test
and mix. 5. Pre-warm Calcium chloride for at least 5
2. Centrifuge at 1500 rpm for 5 minutes. minutes
3. Transfer the plasma into a clean tube. 6. Pipette 25μl plasma into cuvette
4. Turn on the instrument and wait until the 7. Add 25μl aPTT reagent plasma
LED lights up. 8. Incubate for 5 minutes
5. Select “PT” as an active test. 9. Transfer cuvette to measuring area
6. Check calibration. 10. Press “Optic”
7. Pre-warm thromboplastin reagent for at 11. Add 20μl pre-warmed calcium chloride
least 5 minutes and simultaneously start timing
8. Pipette 25µl plasma into a cuvette. 12. Wait for the result and record aPTT
9. Press “Optic” accurately
10. Pre-warm plasma for 1 minute (press 13. Repeat the procedure and report the
timer). average of the tests
11. Transfer cuvette to measuring area 14. Identifies and segregates wastes
12. Add 50µl pre-warmed generated and dispose them properly
thromboplastin/neoplastine
13. Wait for the result. Record.

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[MLS12b] Module 5 CLOT-BASED SCREENING TESTS

PT vs APTT PT APTT TT
Reference Range 12-14 seconds 25-35 seconds 15-20
seconds
Sources of Error 1. Overfilled and underfilled evacuated tubes 1. Sample Collection and Preparation
• Anticoagulant volume must be • Traumatic venipuncture
adjusted if Hct is >55% • Contamination of specimen with
2. Overmixing of tubes tissue thromboplastin
• Inversions should be gentle and only 2. Reagent Preparation
for 3 minutes • Improper storage, water
3. Clotted and visibly hemolyzed samples impurities, incorrection dilution
should not be used 3. Instrumentation
• Hemolyzed samples can shorten the • Flailing light source, fluctuations in
prothrombin time temperature, loss of calibration of
4. Plasma lipemia or icterus can alter the tubing, contamination
results if optical instrumentation is used
• Alerts the optical result of the
specimen
5. Heparin should not be used
• Prolonged prothrombin time
• It should be noted on the lab request
and results if the patient is receiving
heparin therapy
Diagnostic Importance Vitamin K deficiency- malnutrition, Hemophilia A (VIII), B (IX), C (XI)
(Prolonged malabsorption, use of broad-spectrum
antibiotics, newborns

Page 19 of 19