Coagulation: Review and Update for Blood Bankers Donna D. Castellone, MS, MT(ASCP)SH Technical Specialist Department of Special Coagulation New York Presbyterian Hospital/Weill Cornell Medical Center [email protected]

Objectives: Identify basic concepts in coagulation  Analyze testing algorithms  Increase problem solving skills in coagulation  Correlate concept between coagulation and blood bank 

News Flash: When you are busy.. So are we!

What happens when Coagulation meets Blood bank: Fresh Frozen Plasma begins to thaw  Platelets are attracted to one another  Units begin to go out  And you have a marriage made in heaven 

What is Hemostasis? Hemostasis is a system of checks and balances  When the system is activated inappropriately, you will have either a bleed or a thrombotic event  It comprises the vascular system, platelets and a series of enzymatic reaction of the coagulation factors 

Secondary Hemostasis Involves a series of enzymatic reactions  Cascade or waterfall theory  Final result is the formation of a fibrin clot  Includes a system of inhibitors and activators 

Clotting assays: Purpose Screening tests for function of an entire assay or individual components  Utilizes a complex mixture of relatively unstable proteins, difficult to purify  Factors are inactivated, must be activated  Also required activated cofactors, ppl & Ca ions 

Model of Blood Coagulation 1900’s 2 step: conversion of IIIIa by TF  1960’s cascade theory, series of reactions, serine proteases circulate as zymogens  1977: VIIa (TF) can activate IX and X  1980’s: inhibitor of TF identified 

Revised Model: Thrombin generation occurs in 2 phases  Initiation results in a small amount of thrombin  Amplification  Propagation where the bulk of thrombin is formed to promote normal hemostasis 

In Vitro Cascade Allows logical effective lab based screening  Can be evaluated through the PT & APTT  Doesn’t reflect clotting physiologically  Does play a role in laboratory evaluation of a potential clotting disorder 

The Coagulation “Cascade” XII Prekallikrein HMWK

Intrinsic Pathway

XI

XIa IX

Tissue Factor VIIa IXa

Extrinsic Pathway

VIIIa

Xa

X

X Va

Prothrombin

Thrombin

Fibrinogen

Fibrin

Importance of PT & APTT   

Screening tests that provide a tremendous amount of information to the physician Can be performed quickly and accurately Responsibility of the laboratory to ensure results are accurate & reproducible - the basis being the reagents & their sensitivity

What does that mean? Many reagents are insensitive to certain factors  This means you can have an abnormal level of a factor, with a normal PT or APTT  Patients with between 30-40% of factor levels are fine  However, that is not always good enough 

For example: Take assayed pooled normal plasma, make dilutions in deficient plasma – look at IX: PNP Dilution APTT(25.5-35.5) 100% 33.2 75% 750ul + 250ul 33.9 50% 500ul + 500ul 34.2 35% 350ul + 650ul 34.8 20% 200ul + 800ul 35.0 normal 15% 150ul + 850ul 37.0

Solution Know your reagents  They could be your worst nightmare!  Coagulation laboratories should do this even if they don’t do factor assays  Information should go to : hematologists, & blood bank  We do workups on normal PT & APTT’s  Worst factors are: II, IX and XI 

The Prothrombin Time Developed by Quick 1 part human brain thromboplastin 1 part patient plasma 1 part Calcium

Prothrombin Time Test TF/FVIIa

FX

Factors detected by PT FXa/FVa

Prothrombin

Thrombin Fibrinogen

Fibrin

Prolonged Prothrombin Time PT is exclusive for VII  Both PT & APTT prolonged- common pathway - I, II, V & X  Most likely reason, due to oral anticoagulation 

Monitoring Oral Anticoagulation The response is variable & unpredictable  If level is inadequate, increases risk of thrombosis  If level is excessive, increase of a bleed 

Pharmacology Coumadin or warfarin result in the inability of the liver to carboxyl ate the glyutamyle residues of Vitamin K factors  II, VII, IX, X, Protein C and S  Render them non-functional, impairing fibrin formation  Loss of function is 1/2 life dependent  VII is first, II is last 

Goal To maintain a narrow therapeutic range  The recommendation for anticoagulation is: Prothrombin Time = 1.5-2.5 times the normal range 

How? Coumadin can be administered for life  How can you monitor coumadin and enable patients to have freedom?  Method to standardize coumadin administration  Regardless of instrument/reagent/ hospital combination 

Reference Method Used a manual method for performing PT’s  Utilized the Manchester Reagent, or human brain thromboplastin  “Gold Standard” - most sensitive reagent 

ISI- 1.0 Gold StandardHuman Brain Thromboplastin Test 60 patients on a stable dose of coumadin, and 20 nl control patients  Utilize the test thromboplastin and IRP  Graph and compare slope of the line - this is the ISI  Must be done for EACH type of thromboplastin and instrument calibration 

FORMULA ISI INR =

PATIENT’S PT --------------------------------Geometric mean of the PT normal range

Variables of the INR The mean of the normal range  The reagents and their stability  The ISI (International Sensitivity Index) established by the manufacturer  The instruments and their calibration of the ISI 

Problems using the INR: Education  Patient must be on a stable dose of anticoagulant  ISI is specific for instruments 

Lower ISI: Better to assess bleeding potential  More sensitive to factor deficiencies  Less sensitive to heparin  More sensitive to liver disease & vitamin k factors  Wider range of clotting times, allows for finer adjustments in dosages 

Variations in the INR: Critically ill patients INR may vary  Effected by body mass, Vitamin K ingestion, patient diet, & liver function  80 drugs will interfere with coumadin  OAC checked 4-5 times/week, than monthly  Patients are therapeutic 65-80% of the time 

Who didn’t understand this? A hospital decided to change to a more sensitive reagent, a lower ISI  Evaluated and choose the reagent  Placed the order for the new reagent  The person ordering, wrote on the order number on the PO for the more sensitive ISI  This directive got lost in the process 

Who is to blame? The reagent came, and was placed on the instrument  The reagent ran for seven weeks  A physician noticed that a patient’s dosage of coumadin constantly had to be increased  The INR was not therapeutic and the patient was bleeding 

Outcome: 932 patients received overdoses of the medication  5 patients, all in their 80’s and 90’s may have died 

Therapeutic Range STANDARD DOSE: RANGE = 2.0-3.0 Prophylaxis Treatment of Thrombosis PE, MI, HIGH DOSE: 2.5-3.5 Mechanical Heart Valve

How do Blood Bankers use this? 

AABB guidelines use for FFP are: 1. Bleeding or planned invasive or surgical procedure and 1 or more of following: a. PT greater than 1.5 MNR or >17 sec b. APTT > 1.5 MNR or > 49 sec c. Deficiency of II, V, VII, X or XI d. Massive transfusion >10units e. DIC f. TTP or HUS

INR INR >3 but 5 but < 9

No bleeding, but at Risk for a bleed

  

hours INR >9 Vitamin K

Significant risk for bleed

 

reaches warfarin stable INR >3

Re-check INR until with bleeding

by  

Hold warfarin, Give Admit patient to hospital Monitor INR until upper Limit, re institute

 

Hold warfarin, monitor INR until

Reaches upper limit of therapeutic Range Weekly: reduce TWD by 20-50% Recheck INR in 72



12 Guidelines from New York Presbyterian Pharmacy6

Hold warfarin, Vitamin K IV Give plasma, get hematology Consult, INR tested 6hour

Activated Partial Thromboplastin Time VIII, IX, XI, XII Intrinsic Factors

APTT:Principle Phospholipid source: Rabbit brain, cephalin, dehydrated rabbit brain, bovine brain & soybean  Activated by contact, Recalcified plasma in presence of a standard amount of ppl  Intrinsic factors 

FXII/XIIa FXI/XIa-> Factors detected by aPTT FIXa/FVIIIa

FXa/FVa

Prothrombin

FX

Thrombin Fibrinogen

Fibrin

APTT: Purpose Screen patients for bleeding  Factors VIII, IX, XI, XII, I, II, V and X  Lupus Inhibitors  Monitors replacement therapy  Acquired factor deficiencies  Monitors heparin 

APTT More diverse and unstandardized  Depends on type and concentration of ppl, more important then type of contact activator  Different sensitivities for factors  Heparin responsiveness 

Activated Partial Thromboplastin Time - Consists of recalcifying plasma in the presence of a standardized amount of platelet-like phosphatides and an activator of the contact factor - Detects bleeding orders to XII, XI, X, IX, VIII, V, II &I - Will NOT detect VII, XIII or qualitative or quantitative platelet disorders

Prolonged APTT: Check for heparin contamination - best test for residual heparin - Thrombin Time  Repeat on a new sample  Check for presence of a Factor Deficiency or an Inhibitor by performing a mixing study 

Case: APTT = 78.0 seconds  Look at VIII, IX, XI  XII don’t tend to bleed  Results: IX = 95% XI = 102% VIII= 3-7 days Malabsorption: cholestatic liver disease chronic diarrhea celiac disease abeta lipoproteinemia cystic fibrosis

Laboratory anomalies In Vitamin K deficiency: PT-early and more severe  PTT  CBC-normal platelet count unless in DIC  Thrombin time –normal (looks at fibrinogen deficiencies) most sensitive test to determine residual heparin  I:I mix corrects 

Liver disease: Main classes of defects are :  Impaired coagulation  Thrombocytopenia and platelet function  DIC  Systemic fibrinolysis 

Coagulation defects: 

Decrease in hepatic synthesis: all are liver produced except vWF Affect both clotting and fibrinolytic system Quantitative and qualitative defects



Impaired Clearance of activated coagulation factors

Factor anomalies: Factor VII is a sensitive indicator of liver function.  Short t ½- first marker of parenchymal liver disease and Vitamin K deficiency  Unaffected by DIC and inflammation 

Factor anomalies: Factor V remains low in acute and chronic liver disease- decrease in production and Factor consumption  Is elevated in biliary tract disease 

Factor anomalies: Factor VIII is synthesized in liver vascular cells while von Willebrand factor is synthesized in endothelial cells and megakaryocytes.  vWF is elevated in hepatic insufficiency.  Factor VIII becomes reduced in DIC  When we look for liver disease, decreased V and increased VIII  Why not other factors? 

Discussion-Vitamin K therapy 

Vitamin K therapy: Infant/Young child:1-5 mg Older child: 5-10 mg Adult:10 mg



Route: parenteral is recommended subcutaneous 2-6 hr correction intravenous oral 6-8 hr correction

Therapy continued: Fresh frozen plasma/Cryoprecipitate

Dependent on severity  Dose 10-15 ml/kg  Assess coagulation values after infusion+/- factors II,V,VII, Fibrinogen  Consider plasma exchange if fluid overload  Cryoprecipitate if Fibrinogen < 75 mg/dl  Cryoprecipitate contains VIII, vWF,XIII, Fibrinogen 

Platelet support: Indicated for active bleeding  Indicated for counts 150 mg/dl 

The other side of the coin: Liver disease may promote hypercoagulability via:  Increased Factor VIII, V WF  Reduced Protein C,S,Antithrombin  Presence of antibodies: anti phospholipid, anticardiolipin,ANCA in adult diseasepromotion of portal or splenic or deep vein thrombus 

Summary: Liver disease is associated with a wide variety of abnormalities in coagulation system.  Bleeding can be severe.  Complicated by infection, endotoxemia and encephalopathy  Use stepwise approach to therapy to restore balance. 

So why were are the results normal? Should they have been with abnormal screening tests?  Both prolonged Common pathway  Any ideas? 

We tested the transfused unit We test what we get!

Case Study: 25 year old female  Needed to have orthopedic surgery  History of bleeding and bruising  Heavy menses  All coagulation tests are normal  What should we look at? 

Primary Hemostasis 

We’ve got to stick together......

Platelets In Primary Hemostasis

Shear Platelets

Subendothelium

Collagen/vWF Adhesion

Fibrin

Aggregation Coagulation

Platelet Receptor-Ligand Interactions

GPIIb-IIIa GPIa-IIa GPIV GPVI GPIb Collagen

CONTACT

FIB vWF

GPIIb-IIIa vWF

vWF

ADHESION

AGREGGATION / RELEASE

Function Response to injury - undergo a shape change - disc to a spiny sphere  Adhere - as a spiny sphere, they stick to the site of vessel injury. This is primary aggregation and is reversible  Release - platelets release the contents of dense and alpha granules - secondary aggregation, is irreversible 

Function   



Aggregation - in response to chemical changes, events lead to cohesion to other platelets End result is to stabilize the clot Release Factor V and PF3 to accelerate the coagulation cascade and promote the activation of clotting factors. Stabilize platelet plug, with fibrin clot

Platelet Abnormalities: Thrombocytopenia - most common cause of bleeding  Platelets are low in number, nl in function  Patients will bleed, below 50,000  10,000 can cause CNS bleed  ITP, Viral, Drugs 

Platelet Aggregation Aggregation off platelets can be induced by adding various reagents such as ADP, EPI, Collagen, Ristocetin, Thrombin & Aracadonic acid  Using PRP sample is placed into an aggregometer, change in optical density as platelets aggregate 

Platelet Disorders    

Bernard Soulier-giant plts-lacks GpIb & can’t adhere to surface of the cell Thrombastenia - severe mucous membraneLacks GpIIb/IIIa Storage pool-no granules, no release, mild bleed Cyclo-oxygenase , Hermanskly-Pudlak, Wisckott-Aldrich, May-Hegglin

Results: Patient had no secondary response to ADP, EPI, decreased response to thrombin  Storage pool disease  Now blood bank takes over  Need to give her good platelets to get through the surgery 

Real life - you can’t make this stuff up!

Coagulation is :