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

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 

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