Domain Structure of Clotting Factor VIII (FVIII)

Synthesis as Peptide Chain

•   FVIII is synthesized as a single chain polypeptide of 2351 amino acids
• A 19-amino acid signal peptide is cleaved by a protease shortly after synthesis

Arrangement of single chain of FVIII

• Domain structure of FVIII is as under:
• A1 – a1 – A2 – a2 – B – a3 – A3 – C1 – C2
• FVIII amino acid sequence suggests that the molecule is composed of
• A triplicated homology region
• A1 domain
• A2 domain
• A3 domain
• A duplicated homology region
• C1 domain
• C2 domain
• Heavily glycosylated B domain  
• A1 domain
• Cleavage site (Arg336/337 & 562/563) by APC
• FVIII binds with
• Phospholipids via C1 & C2 domains
• VWF via a3 domain

Significance & Physiological Role of Tissue Factor Pathway Inhibitor (TFPI)

• Tissue Factor Pathway Inhibitor (TFPI) is located

• On Endothelial Cells (ECs)
• On Monocytes
• In Platelets 
• In plasma 

• Bulk of TFPI is associated with ECs

• Measurement of low TFPI in plasma doesn’t mean TFPI deficiency 
• TFPI associated with ECs → 4X ↑in TFPI upon Heparin injection
• Most of circulating TFPI is bound to lipoproteins & the C terminals are variably degraded  

• Physiological Role 

• TFPI inhibits FVIIa in a FXa-dependent manner
• TFPI binds to & directly inhibits TF-FVIIa-FXa complex → So, it effectively halts TF initiated activation of coagulation but only after sufficient FXa has been generated to propagate coagulation
• Plasma TFPI has reduced anticoagulant activity
• So, likely not very important anticoagulant role in vivo

• Therapeutic Role 

• TFPI binds to Heparan Sulphate on EC surface
• UFH or LMWH displace this bound TFPI → This released TFPI contributes to antithrombotic activities of these drugs

• Isoforms of TFPI

• Two isoforms exist 
• TFPIα
• TFPIβ 
• TFPIα has 3 Kunitz type protease inhibitory domains → K1, K2, K3 
• K1 domain inhibits FVIIa in a FXa dependent manner 
• K2 directly binds & inhibits FXa
• Protein S (PS) acts as cofactor in the formation of TFPI-FXa complex

Physiological Inhibitors of Coagulation System

There are many physiological inhibitors impacting various stages of coagulation system. Most important inhibitors with known physiology are enumerated as under: 

• Antithrombin 
• Protein C
• Protein S 
• Heparin
• Heparin cofactor II
• TFPI (Kunitz type inhibitor)
• PZ & PZ dependent inhibitor 
• 𝛂1-Antitrypsin
• 𝛂2-Antiplasmin
• α2-Macroglobulin
• Protease Nexin 2 
• C1-esterase inhibitor

Afibrinogenemia & Important Manifestations/Complications

• Afibrinogenemia is an inherited Quantitative Autosomal Recessive disorder 

• Plasma and platelet levels of the fibrinogen are not measurable 
• Plasma levels <0.1 g/L or <10 mg/dL

• Homozygous or Compound heterozygous

• FGA gene is most commonly involved

• Clinical manifestations

• Bleeding

• Neonatal bleedings like umbilical stump bleeding

• Gastrointestinal bleeding

• Urological bleeding 

• Central nervous sytsem bleeding

• Spleen (with splenic rupture)

• Abnormal wound healing

• Obstetric complications

• 1^st trimester fetal loss

• Antepartum hemorrhage

• Postpartum hemorrhage

• Spontaneous abortion

• Placental abruption

• Bone cysts

• Mostly in long bones → possible effects of bleeding

NOTE: Arterial or venous thrombosis is possible 

Disorders of Fibrinogen (Clotting Factor I)

Quantitative Disorders 

o   Inherited

§  Autosomal Recessive Afibrinogenemia

§  Autosomal Dominant Hypofibrinogenemia 

o   Acquire

§  Hypofibrinogenemia

§  Hyperfibrinogenemia

Qualitative Disorders 

o   Inherited

§  Autosomal Dominant Dysfibrinogenemia

§  Autosomal Dominant Hypodysfibrinogenemia

o   Acquired

§  Liver disease

§  Malignancies 

§  Antifibrinogen antibodies 

Management Principles of Subtherapeutic INR

• Patient education regarding 
• Compliance

• Drug/Food interactions with warfarin  

• Effects of low INR 

• When truly genetic resistance 

• Increase the dose of warfarin 

• Titrate the dose carefully to >100 mg/day with regular monitoring of INR 

• Change the anticoagulant 

• UFH, LMWH, Fondaparinux 

• Rivaroxaban, Dabigatran 

• Other Vitamin K antagonists 

• Bishydroxycoumarin

• Phenprocoumon 

• Acenocoumarol

• Phenindione

Investigating Subtherapeutic & Supratherapeutic INR

•  Full history regarding 

• Compliance
• Drugs
• Diet
• Associated medical conditions 

•  Plasma levels of Warfarin 

• Therapeutic levels 
• At specific intervals after administration 

• FII & FX assays 

• Pharmacogenetic analysis 

• P450 CYP2C9*2 and CYP2C9*3 polymorphism →↑ INR
• Missense mutations of VKORC1 gene CYP 2D6 & CYP2A6 →↓ INR
• Polymorphism in VKORC1 gene → Variable INR 

Causes of Supratherapeutic INR despite Full-Dose Warfarin Administration

 Hereditary

• Polymorphisms in P450 CYP2C9*2 and CYP2C9* → ↓ metabolism → ↑ Warfarin t_1/2

  Acquired

• Non-compliant patient 
• Drugs

Acetaminophen	Cephalosporins	Tramadol
Allopurinol	Fluoroquinolones	Capecitabine
Amiodarone	Macrolides (Clarithromycin, Erythromycin)	5-FU
Androgens	Penicillin except Nafcillin, Dicloxacillin	Imatinib
Methyldopa	Trimethoprim-Sulfamethoxazole	Cholesterol lowering drugs less Cholestyramine
Testosterone	Metronidazole	Cimetidine
Oxandrolone	Fluconazole	SSRIs
Methyltestosterone	Voriconazole	Sitaxentan
Tamoxifen	Miconazole (oral)	PPIs
Glucosamine	Clofibrate	Sulfinpyrazone

Causes of Subtherapeutic INR despite Full-Dose Warfarin Administration

• Hereditary 
• Mutations of VKORC1 Gene → Warfarin resistance 
• Duplication or multiplication of Cyt P450 enzyme genes (P450 CYP2D6 & CYP2A6)
• ↑ metabolic clearance of Warfarin

• Acquired 
• Poor patient compliance 
• High consumption of Vitamin K
• Decreased absorption of Warfarin 
• Gastroenteritis
• Coeliac disease
• Chronic pancreatitis
• Short gut syndrome
• Increased clearance of Warfarin 
• Hypoalbuminemia → ↑ free fraction of Warfarin → ↑ clearance and ↓ plasma half-life
• Hyperalbuminemia → Paradoxical effect by increased binding of Warfarin 
• Hyperlipidemia 
• ↑ Lipids → ↑ Vitamin K pool → Warfarin resistance 
• Drugs decreasing the effect of Warfarin 
• Azathioprine
• Barbiturates 
• Carbamazepine 
• Cholestyramine
• Corticosteroids 
• Dicloxacillin & Nafcillin 
• Estrogen preparations
• Spironolactone → ↓ Plasma volume → ↑ Coagulation factor levels  
• Griseofulvin 
• Rifampicin 
• Contraceptive pill  
• Ritonavir
• Saint John's wort
• Sucralfate
• Tobacco smoking 
• Powerful inducer of P450 (CYP1A1 & CYP1A2)
• Tobacco leaves are rich in Phylloquinone (vitamin K1)
• Each gram of Tobacco → 50 μg Vitamin K1 (exceeds the amount found in food sources) 

Genetic Changes Affecting the Pharmacokinetics of Warfarin Dosing

• Polymorphisms in P450 CYP2C9*2 and CYP2C9*3 Hepatic Microsomal Enzyme System

• ↓ metabolism of warfarin

• ↓ warfarin dose requirements

• ↑ INR levels at a given dose

• ↑ risk for bleeding during warfarin therapy

• Duplication or Multiplication of Cyt P450 Enzyme Genes (P450 CYP2D6 & CYP2A6 )

• ↑ metabolic clearance of Warfarin 

• Subtherapeutic INR 

• Missense mutations of VKORC1 gene

• Warfarin resistance

• Polymorphisms in VKORC1 gene

• Inter-individual variability in the dose-anticoagulant effect of warfarin

Major Constituents of Alpha (α) Granules of Platelets

• α-granules are one of 4 types of granules in Platelets
• The other 3 include → Dense granules, Lysosomes & Peroxisomes 
• There are approximately 80 α-granules per platelet

Major Constituents of  α-granules are as per the following table:

Physiological role	Constituents of α-granules
For platelet aggregation	VWF, FI, Fibronectin
For irreversible aggregation	Thrombospondin
For Coagulation/anticoagulation	FI, FV, FXI, TAFI, AT, PS
For fibrinolysis	PGL, PAI-1, α2-antiplasmin
Membrane proteins	GPIIb/IIIa, P-selectin, CD40L
EC activation	TGF-β
For leukocyte recruitment	PH4, β-Thromboglobulin, ENA78, SDF-1α, CCL5
Growth factors	EGF, FGF, HFH, IGH-1, PDGF
For angiogenesis/inhibition	VEGF-A, VEGF-C, PDGF, Endostatin, Angiostatin
Bacterial killing	Microbial proteins
Immune molecules	IgG, C3 & Cd precursors, C1 inhibitor, Factor H
Proteases	Protease Nexin II
Matrix breakdown	MMP-2, MMP-9
Others	Amyloid β-protein precursor, Gas6

INR Calculation for Patients with Liver Failure/Disease

• Routine method of calculating INR by derived ISI value of a particular Thromboplastin regents cannot be used for the patients with liver disease.

• This is because the variables that affect the INR in liver disease are different from those on oral anticoagulants (Warfarin).

• In routine testing and monitoring of patients with liver disease, instead of INR, only PT value should be used.

To use INR in Patients with Liver Disease 

• Thromboplastin needs to be calibrated for ISI^Liver using Citrated PPP samples from patients with liver cirrhosis 

• This ISI^Liver can then be used to calculate INR in patients with liver disease 

International Sensitivity Index (ISI) value & Calibration of Test Thromboplastin Reagent

International Sensitivity Index (ISI)

• Thromboplastins are derived from different sources (human, rabbit etc.) for use in Prothrombin Test (PT).
• The PT carried out with each of these Thromboplastins gives different results → Different sensitivities to recognize clotting factor deficiency or coagulation system.
• This difference in sensitivity of Thromboplastins to recognize the status of coagulation system under investigation is called Sensitivity Index.   
• International WHO Reference Thromboplastin has been assigned International Sensitivity Index value of 1.0 
• All other reference thromboplastins are calibrated to this above mentioned WHO Reference Thromboplastin & an ISI value is assigned to that particular thromboplastin in comparison.  
• These Reference Thromboplastins (calibrated against WHO Reference Thromboplastin) are in turn used to calibrate a Test Thromboplastin (any Thromboplastin made in lab or available commercially). 
• Special Note: A test thromboplastin must be calibrated against a reference thromboplastin from the same species. So, Rabbit Thromboplastin is calibrated against Rabbit Reference Thromboplastin. 

Method of Calibrating a Test Thromboplastin

• Step 1 
• Get Citrated Platelet Poor Plasma (PPP) from 20 healthy donors & from 60 patients stabilized on Tablet Warfarin for ≥6 weeks. 
• Step 2 
• Perform Prothrombin Test (PT) using Reference Thromboplastin on above mentioned 80 (20 + 60) samples in duplicate & take mean PT value for each 
• Total tests with reference thromboplastin → 80 x 2 = 160
• The maximum allowable difference between the two reading on same sample during duplicate testing is only 10%
• If difference is >10% → Repeat PT on that sample in duplicate again
• Step 3
• Perform PT using Test Thromboplastin on above mentioned 80 (20 + 60) samples in duplicate & take mean PT value for each
• Total tests with test thromboplastin → 80 x 2 = 160 
• The maximum allowable difference between the two reading on same sample during duplicate testing is only 10%
• If difference is >10% → Repeat PT on that sample in duplicate again
• Step 4
• The means of each pair of readings for Reference Thromboplastin (Step 2) & Test Thromboplastin (Step 3) are plotted on a Log-Log Graph:
• Y-axis → Mean PT readings for Reference Thromboplastin 
• X-axis → Mean PT readings for Test Thromboplastin 
• Step 5
• Draw the Line of Best Fit (on computer, simple linear regression may be applied instead – sufficiently accurate)
• Step 6
• Mark points on the best fit line as under: 
• Point A → Just below the lowest recorded PT
• Point B → Just above the longest recorded PT
• Draw a horizontal line from Point A parallel to X-axis 
• Draw a vertical line parallel to Y-axis
• Mark the point where the two lines intersect as Point C
• Step 7 
• Measure the lengths of the lines accurately in mm (millimeters)
• Calculate the Slope as under: 
• Slope = Vertical line meeting Point C / Horizontal line meeting Point C 
• Slope =  (Point B to Point C (mm))/(Point A to C (mm))
• Example: if B – C = 55 mm & A – C = 35 mm 
• Slope = 55/35 = 1.57 
• Step 8 
• Multiply the Slope value with the ISI of Reference Plasma 
• Example: 
• If the ISI of Reference Plasma is 1.1 
• ISI of Test Thromboplastin = 1.1 x 1.57 = 1.74 
• Step 9 
• Calculate the International Normalized Ratio (INR) from the above calculated ISI value
• INR is the ratio of Patient’s PT compared to Geometric Mean Normal PT (GMNPT) that is corrected for the sensitivity of the Thromboplastin used 
• GMNPT → Geometric Logarithmic Mean of Normal PT that is determined for each batch of Thromboplastin by testing 20 normal PPP samples 
• PT Ratio = (PT of the Patient)/GMNPT
• INR =[PT Ratio]^ISI

Selection of PT Reagent (Thromboplastin) for Coagulation Lab

• Selection of Thromboplastin from a variety of available/offered Thromboplastin reagents is very important as the therapeutic monitoring of Warfarin depends upon INR. 
• Single most important criterion for selection of Thromboplastin is Thromboplastin reagent with value of ISI closer to 1.0

Impact of Thromboplastin with high ISI value 

• Small change in PT would translate into a large change in INR 
• Large change in degree of anticoagulation or coagulation factor deficiency  
• So, when a small change in dose or physiology of patient occurs → Therapeutic intervention becomes necessary for physician
• Analysis becomes imprecise 
• Coefficient of variation (CV) varies with the ISI value  
• When the clinical decision is based on Target PT ratio, the range of PT ratio becomes small for any given value of INR 

Selection of APTT Reagent for Coagulation Lab

• Selection of Activated Partial Thromboplastin Test (APTT) reagent from a variety of available/offered reagents is very important. 
• Selected APTT reagent must be sensitive to: 
• Deficiency of FVIII → ≤35 – 40% (35 – 40 U/dL or 0.35 – 0.4 IU/mL) concentration of FVIII
• Deficiency of FIX → ≤35 – 40% (35 – 40 U/dL or 0.35 – 0.4 IU/mL) concentration of FIX
• Deficiency of FXI → ≤35 – 40% (35 – 40 U/dL or 0.35 – 0.4 IU/mL) concentration of FXI
• Unfractionated Heparin (UFH) over therapeutic range of 0.3 – 0.7 anti-Xa IU/mL
• Lupus anticoagulant (optional as per requirement of the lab setup)

Performance of Activated Partial Thromboplastin Time (APTT) Test in Coagulation Lab

 Requirements

• Personal protective equipment
• Lab coat 
• Protective Apron 
• Lab-grade gloves 
• Shoe covers
• Protective face shield or protective goggles 
• Spill kit 
• Workstation 
• Clean & disinfected workstation 
• Tube discarder box 
• Sharps discarder 
• Calibrated Pipettes
• Calibrated water-bath 
• Coagulation calibrated electrical centrifuge 
• Thermometer 
• Optimally functioning digital stopwatch
• Labelling marker 
• Light source (Bright lamp)
• PT result recording sheet 
• Calculator 
• Test related 
• Multiple clean glass tubes 
• Citrated Control Platelet Poor Plasma (C-PPP)
• Citrated Test/Patient's Platelet Poor Plasma (T-PPP)
• QC verified Thromboplastin Reagent 
• QC Verified Calcium Chloride CaCl₂ Reagent 

Procedure 

• Step 1 
• Label a clean glass test tube as CaCl₂
• Add >100 μL CaCl₂ reagent in this tube
• Place this test tube in water-bath at 37 ⁰C for >2 mins 
• Step 2 
• Label 4 clean glass test tubes as following: 
• C1 → Citrated Control PPP
• C2 → Citrated Duplicate Control PPP
• T1 → Citrated Test PPP
• T2 → Citrated Duplicate Test PPP
• Step 3 
• Add 100 μL Citrated Platelet Poor Plasma (PPP) into C1 labelled tube 
• Incubate C1 labelled tube in water-bath at 37⁰C for 02 mins
• Step 4 
• Take out 100 μL APTT reagent (from commercial APTT reagent bottle at room temperature) and add it to C1 labelled tube (which has completed 2 mins incubation in water-bath in Step 3)
• Incubate C1 labelled tube for 3 more minutes in water-bath at 37⁰C
• Step 5
• After completion of 3 mins incubation in step 4, take out 100 μL CaCl₂ reagent from CaCl₂ labelled tube (which is already in water-bath from step 1) and add it to C1 labelled tube
• Start the stopwatch 
• Step 6
• From 25 seconds onwards remove the C1 labelled tube from water-bath to see for clot formation at regular intervals (every 2 seconds)
• Note the time when the clot has formed & stop the Stopwatch
• Step 7
• Repeat the procedure as per above steps in C2 labelled tube 
• Take the average of the readings from C1 & C2 labelled tubes
• Step 8
• Now, repeat all the above mentioned steps in duplicate with Test PPP from the patient in T1 & T2 labelled tubes
• Record the average of the readings from T1 & T2 labelled tubes

Reporting of Results 

• Results are reported as per following two formats:
• The mean (average) of the APTT readings run in duplicate in seconds
• APTT ratio between Citrated Test & Control PPP
• For APTT Value of Citrated Control PPP in Routine 
• GMNAPTT is calculated as the mean of PT for 20 healthy individuals as under:
• 10 samples from healthy males 
• 10 samples from healthy females
• No sample from those receiving any oral anticoagulant

Interpretation of Results

• Causes of Isolated APTT prolongation with normal PT (Click For Details)
• Factor VIII, IX, XI, XII, Prekallikrein or HMWK deficiency
• FVIII deficiency secondary to VWD
• Circulating anticoagulant, e.g., Lupus Anticoagulant
• Heparin or direct acting anticoagulant
• Mild factor II, V or X deficiency

Performance of Prothrombin Time (PT) Test in Coagulation Lab

Requirements

• Personal protective equipment
• Lab coat 
• Protective Apron 
• Lab-grade gloves 
• Shoe covers
• Protective face shield or protective goggles 
• Spill kit 
• Workstation 
• Clean & disinfected workstation 
• Tube discarder box 
• Sharps discarder 
• Calibrated Pipettes 
• Calibrated water-bath 
• Coagulation calibrated electrical centrifuge 
• Thermometer 
• Optimally functioning digital stopwatch
• Labelling marker 
• Light source (Bright lamp)
• PT result recording sheet 
• Calculator 
• Test related 
• Multiple clean glass tubes 
• Citrated Control Platelet Poor Plasma (C-PPP)
• Citrated Test/Patient's Platelet Poor Plasma (T-PPP)
• QC verified Thromboplastin Reagent 

Procedure 

• Step 1 
• Label a clean glass test tube as Thromboplastin 
• Add >200 μL Thromboplastin reagent in this tube
• Place this test tube in water-bath at 37 ⁰C
• Step 2 
• Label 4 clean glass test tubes as following: 
• C1 → Citrated Control PPP
• C2 → Citrated Duplicate Control PPP
• T1 → Citrated Test PPP
• T2 → Citrated Duplicate Test PPP
• Step 3 
• Take C1 labelled tube & add 100 μL Citrated Platelet Poor Plasma (PPP) into it
• Incubate C1 labelled tube in water-bath at 37⁰C for 02 mins
• Step 4 
• Take out 200 μL 37⁰C pre-incubated Thromboplastin reagent (from Thromboplastin Labelled Tube) and add it to C1 labelled tube (which has completed 2 mins incubation in water-bath in Step 3)
• Immediately start the Digital Stopwatch 
• Step 5
• From 9 seconds onwards remove the C1 labelled tube from water-bath to see for clot formation at regular intervals (every 2 seconds)
• Note the time when the clot has formed & stop the Stopwatch
• Step 6
• Repeat the procedure as per above steps in C2 labelled tube 
• Take the average of the readings from C1 & C2 labelled tubes
• Step 7
• Now, repeat all the above mentioned steps in duplicate with Test PPP from the patient in T1 & T2 labelled tubes
• Record the average of the readings from T1 & T2 labelled tubes

Reporting of Results 

• Results are reported as per following two formats:
• The mean (average) of the PT readings run in duplicate in seconds
•  PT ratio between Citrated Test & Control PPP
• For PT Value of Citrated Control PPP in Routine 
• GMNPT is calculated as the mean of PT for 20 healthy individuals as under:
• 10 samples from healthy males 
• 10 samples from healthy females
• No sample from those receiving any oral anticoagulant
• GMNPT is calculated for every new Thromboplastin reagent

Interpretation of Results

• Causes of Isolated PT prolongation with normal APTT (Click For Details)
• FVII deficiency
• Start of oral anticoagulants (Warfarin)
• Lupus anticoagulant: Some thromboplastins are sensitive to LA
• Mild liver impairment or vitamin K deficiency
• Depending upon reagent used mild deficiency of II, V, X
• Causes of Isolated Shortening of PT
• Following treatment with rFVIIa (NovoSeven or SevenFact)

Difference Between Anti-Thrombotic and Anticoagulant Agent

• Antithrombotic Agents

• A group of drugs that include the following:
• Antiplatelet drugs
• Anticoagulants
• Anticoagulant Agents

• A variety of agents that inhibit one or more steps in the coagulation cascade
• The following groups of drugs are included:
• Unfractionated Heparin
• Low molecular weight heparins
• Fondaparinux
• Vitamin K Antagonists
• Direct FXa inhibotors
• Indirect FXa inhibitor
• Direct FIIa inhibitors
• Indirect FIIa Inhibitors
• Investigative Anticoagulants
• FXIa inhibitors (Milvexian, Asundexian)
• FXI inhibitors (Abelacimab)
• Monoclonal Antibody for Active site of FXIa (Osocimab)
• FXI synthesis inhibitor (Fesomersen)
• FXIIa inhibitor (AB023)
• Allosteric inhibitor of FXIa (Sulfated chiro-inositol)
• TF/factor VIIa inhibitors (Recombinant TFPI and anti-TF antibodies, Nematode anticoagulant peptide-2)
• Factor VIII inhibitor (TB-402)
• Throombomodulin (ART-123)
• Factor IXa inhibitor (REG1)
• Factor XIIa inhibitor (Infestin-4)
• Protein disulfide isomerase inhibitors (quercetins)
• Polyphosphate inhibitors

Factors Potentially Affecting Prothrombin Time (PT)

Factors potentially affecting or influencing PT may include the following:

• Shortening of PT

• Physiological
• Pregnancy
• Aging
• Exercise
• Pathological
• VTE & ATE
• Malignancy
• Sepsis
• Thyroid disorders
• Therapeutic
• rFVIIa
• Estrogen medications
• Spurious
• Prolonged venous stasis of >3 mins
• Coagulation before centrifugation
• Hyperbilirubinemia
• Hypertriglyceridemia
• Hemolyzed sample
• Excess vigorous mixing of blood
• Chilling in refrigerator or placing on ice
• Incomplete Platelet separation
• Sample re-run after freezing
• Tube leaded into long track/belt in completely automated system
• Analytical error

• Prolongation of PT

• Physiological
• None
• Pathological
• Vitamin K deficiency
• Liver Disease
• FI, FII, FVII, FV, FX deficiency
• Afibrinogenemia
• Dysfibrinogenemia
• DIC
• Lupus anticoagulant
• Therapeutic
• Warfarin & other VKAs
• Direct FIIa inhibitors
• Oral → Dabigatran
• Parenteral
• Hirudin
• Bivalirudin
• Argatroban
• Anti-FXa drugs
• Heparin, Heparinoids, LMWH, Pentasaccharides
• Spurious
• Heparin contamination
• Daptomycin therapy
• Clotted sample
• EDTA contamination (calcium chelation)
• Under filled tube (<90% of the nominal volume)
• Failure to mix specimen after collection
• Sample held too long before analysis
• Sample placed on wrong temperature
• Use of centrifuge brake
• Analytical error
• Unfractionated Heparin
• Most reagents are less sensitive to UFH due to
• Nature of PT assay
• Inclusion of Heparin Neutralizing agent in most PT reagents
• PT reagent is sensitive when concentrated Heparin contaminates the sample
• LMWH
• PT reagents are even less sensitive to LMWH than WFH
• Heparinoids (anti-FXa activity)
• PT reagents are even less sensitive to Heparinoids than WFH
• Pentasaccharides (Fondaparinux)
• PT reagents are even less sensitive to Fondaparinux than WFH
• Clotted Sample
• Serum or fully Clotted sample → No fibrinogen → No clot in PT, APTT or TT
• Partially clotted sample → Falsely Prolonged PT, APTT & TT or Shortened APTT
• Hemolyzed sample
• Hemolyzed RBCs → Phospholipid membranes → Coagulation accelerated → Shortened PT
• Hemolyzed RBCs → Platelet activation → Impaired PT results
• Change in the color of plasma & particulate material → interferes with optical end-point detection methods
• Over & underfilled sample tube
• Lupus anticoagulant
• Most PT reagents are insensitive to LA
• Some PT reagents are sensitive to LA

• Daptomycin therapy
• Spuriously prolongs the PT
• Depends upon 2 factors
• Thromboplastin source
• Especially prolonged PT when recombinant human or rabbit thromboplastins are used
• Phospholipid type of the PT reagent
• Prolongation occurs mostly when Phosphatidylglycerol is used as phospholipid in PT reagent

• Vigorous shaking mixing of blood sample
• Hemolysis & platelet activation → activation of coagulation cascade → PT falsely shortened
• Chilling in refrigerator (≤4⁰C) or placing the plasma on ice
• Activation of FVII activation of coagulation cascade → PT falsely shortened 

Causes of Isolated Prolongation of Thrombin Time (PT)

 Isolated prolongation of PT with normal APTT is observed in following conditions: