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
with no bleeding
Day 1: subtract 5-10%
total
Weekly dose (TWD)
Weekly reduce TWD in 72
hours
INR 4.0-5.0
Day 1: no
warfarin
Weekly reduce TWD by 1020%

INR >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= <1%

Known Hemophiliac
Bleeding
 Needs to see if patient has an VIII inhibitor
 Bethesda Assay
 Dilutions of patient plasma, buffer & PNP
 Looks at the amount of inhibitor present
that will inhibit 50% of the factor

Results








Bethesda unit= 153% - too high to give
cryoprecipitate
How to treat?
High titer – bypass cascade
Used in immune tolerance induction
rVIIa- 90ug/kg, short ½ life 2-3 hours
Can not monitor in laboratory
PT too short to measure
Increased risk of thrombosis
OTHER
Activated Prothrombin Complex
 FIBA, II, VII, IX and X
 Made from blood
 Increased risk of disease & thrombosis
 Autoantibodies: corticosteroids and
cytotoxic agents to suppress formation
 RITUXIMAB-monoclonal B cell antibodies

Case
67 year old male hernia repair
 Normal coagulation & hematology tests
 Patient comes in FU/1 week post op
 Platelet count 30,000
 Notes swelling in leg
 Admits into hospital
 Next day platelet count 15,000
 Resident orders platelets

CLUES:
Swelling in leg
 Over zealous resident
 No bleeding
 Afraid of CNS bleed
 Are platelets really being destroyed?
 ITP?

Heparin Induced
Thrombocytopenia
Occur 5-7 days after administration of
heparin
 IgG antibodies are formed
 Cause irreversible skin necrosis, limb
amputation, high mortality risk 50-80%
 Will see decrease in platelets - about 50%
drop/day
 Will thrombosis -”White Clot Syndrome”

Type I HIT
Modest decline in platelets
 Cause is unknown
 Doesn’t involve immune/antibody complex
 Possible effect of heparin on platelets
 Results in increased platelet sequestion &
consumption
 Remove patient from heparin

Type II HIT
Occurs 5-14 days after heparin (unless
previous exposure)
 Risk for thromboembolic complications
 Immune origin
 Increased megakarocytes in Bone Marrow
 Decreased platelet survival
 IgG antibodies

Case Study
A true marriage of
Hematology & Coagulation
Medical history:14 yr old female
May 2000-diagnosis of pre B cell ALL.
Presented with ecchymoses and
hepatosplenomegaly.
 August 2003-relapse with
thrombocytopenia. Complication of focal
seizures on ifosfamide.
 Documented cholelithiasis.

Case: 14yr old female
Jan 2004-Maternal stem cell transplant.
 NO GVHD
 Developed CMV antigenemia
 Developed severe hemolytic anemiatreated with IVIG, Rituximab and
Vincristine.
 August 2004- Underwent splenectomy

Past history cont’d:
Developed catheter related Left upper limb
venous thrombus
 Sept 2004-Hemolytic transfusion reaction.
 5 days later presented with extreme
hyperbilirubinemia TB: 66 Direct:52
 Total 140 units of PRBC had been
transfused since Jan 2004.

Laboratory evaluation:
Admission Labs:
 CBC: WBC 11.1 HB 10.4 PLTS 408
 MCV 91.9 MCH 32.4 RDW 15.4MPV 11.9
 N 24 L 30 Bands 8 M 23


PT 20.9 PTT 73.5 INR 3.10
Factor evaluation 10/15:









II
73
V
79
VII
126
VIII
238
X
118
XI
23
Fibrinogen
500
Protein C 88
Protein S 128
Empiric Vitamin K:

Repeat PT/PTT on Vitamin K
Pre
10/15: PT 20.9 PTT 73.5 INR 3.10
10/17: PT 28.8 PTT 128 INR 5.73
Post
10/19: PT 14.1 PTT 35.6 INR 1.45
Discussion-Heparin contamination:





Prolonged PTT and Thrombin time
Heparinase- Hepzyme removes heparin from
plasma.
Other options for neutralization: Protamine,
Polybrene, Anion exchange resins
Reptilase time to confirm Thrombin time is not
affected by Heparin
Volume from indwelling line should be 5 times
dead space.
Risk factors:
Liver disease
 Vitamin K deficiency –prolonged
antibiotics,poor nutritional status.
 Renal dysfunction

Vitamin K deficiency etiologies:

Older child:
Fasting >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 <50-75 K
 Post infusion counts- note recovery
impaired in splenomegaly
 PRBC for HCT <30%
 DDAVP 0.3 ug/kg may shorten bleeding
time

Other Therapy:

Prothrombin complex concentrates:
Life threatening or intracranial bleed
Contains: II, IX, X, variable VII
Lacks V
Risk of thrombosis
Dose 50 units/kg
Principles:
Maintain platelets >50K
 DDAVP to enhance platelet function
 FFP replaces factors
+/- rh Factor VIIa to avoid fluid overload
 Maintain fibrinogen >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 :