COAGULATION:REVIEW & LAB TECHNIQUES
Renee Wilkins, Ph.D., MLS(ASCP)CM
MLS 322 Clinical Hematology
School of Health Related Professions
University of Mississippi Medical Center
What is hemostasis?
The process in circulation where the blood is maintained within the blood vessels
Depends on the balance ofVascularPlasma coagulation factorsPlateletsFibrinolytic system
Primary Hemostasis
PLATELETS
Hemostasis Primary hemostasis
Blood vessel constricts (vasoconstriction)Platelets adhere and aggregate to damaged area
of the vessel wallResults in the formation of the platelet plugPlatelets must be adequate in number and must
be functioning normally in order for the platelet plug to form○ Thrombocytopenia○ Aspirin therapy
Platelets (thrombocytes) Cellular fragments of
megakaryocytes that contain granules
Circulate 9-12 days 2-4 μm Normal platelet count is 150-400,000/μL 100,000 are needed to perform platelet function
testing Thrombocytopenia is the most common cause of
abnormal hemostasis
Platelet
Alpha granules within the organelle contain platelet derived growth factor, platelet factor 4, FV, FXIII, fibrinogen, vWF
Formation of platelet plug Adhesion (1-2 seconds)
Platelets adhere to collagen fibers and vWF to form a bridge (glycoprotein Ib)
Activation Morphological and functional changes Increases internal Ca++, swelling occurs Glycoprotein IIb/IIIa is activated
Aggregation (10-20 seconds) Vasoactive substances like ADP and thrombozane A2 are released along
with platelet procoagulants gp IIb/IIIa is exposed and binds to fibrinogen Other platelets are stimulated to adhere and aggregate When activation and primary adhesion have been achieved, platelets
commence discharge of granule contents. Granule contents include ADP, adenosine triphosphate (ATP), serotonin, calcium, vWF, Factor V, and fibrinogen
Once the plug is formed (1-3 minutes), the platelet mass consolidates to a dense thrombus (3-5 minutes), then retracts and stabilizes (5-10 min)
Plateletglycoprotein Ib
blood vessel wall/subendothelium
von Willebrand factor
collagen
gp IIb/IIIa
fibrinogengp IIb/IIIa
platelet
Primary platelet plug
fibrinogen
platelets
Platelets in coagulation
Platelet factor 3 (PF3) is a phospholipid that resides on or within the platelet
PF3 is required in the activation of certain coagulation factors (to help form thrombin)
Diseases of Primary Hemostasis Bleeds from skin or mucous membranes Factor abnormalities are usually internal Disorders of the vascular system
Abnormalities or damage of lining of blood vessels or subendothelial structures
Superficial bleeding (bruises, petechiae)Platelet count and factors are normal
Platelet disordersThrombocytopenia (<50x109/L)
○ Asymptomatic to severeThrombocytosis (>450x109/L)
Diseases of Primary Hemostasis Inherited (functional disorders)
von Willebrand Disease (vWD) [adhesion]Bernard-Soulier syndrome (adhesion)Glanzmann Thrombasthenia (aggregation)May Hegglin anomaly (quantitative)Disorders of pregnancy (quantitative)
AquiredIdiopathic thrombocytopenic purpura (ITP)Drug-induced platelet dysfunction
○ Aspirin induced platelet dysfunction○ Aspirin resistance
von Willebrand DiseaseInherited, up to 3% of U.S. populationQualitative or quantitative abnormality of von Willebrand
Factor (vWF) – fails to bridge gap (NOT a platelet disorder)vWF is a cofactor to Factor VIII (VIII:vWF complex)Excessively heavy or prolonged menstrual bleeding occurs
in 20% of women○ vWD may be the cause of menorrhagia in as many as 20% of these
women
Other symptoms include easy bruising and gingival bleeding
3 types with Type 2 containing 4 subtypes
Nature of vWF The vWF molecule (protein) is a chain of
identical subunits called multimers vWF is synthesized in alpha granules of
megakaryocytesPrimary hemostasis: vWF bridges GPIb/IX on
platelets and collagen to form the plugSecondary hemostasis: vWF binds to FVIII which
ultimately leads to fibrin formation vWF in plasma varies in size, but most binds
with VIII in plasma (1:1)FVIII is formed in liver before release
vWD
Normal Platelet
glycoprotein Ib
blood vessel wall
No von Willebrand factor means no adhesion… von Willebrand disease
On the flip side….
Giant Platelet
No glycoprotein Ib means no adhesion…. Bernard-Soulier
blood vessel wall
von Willebrand factor
Bernard-Soulier syndrome A.k.a. giant platelet syndrome Rare autosomal recessive disorder <1 in 1,000,000 Moderate to severe thrombocytopenia Abnormal platelet function due to lack of
GPIb/IX complex on platelet Homozygous individuals have lifelong
bleeding tendency; heterozygous have not significant bleeding
Glanzmann Thrombasthenia Rare deficiency of glycoprotein IIb/IIIa on
platelet gp IIb/IIIa binds fibrinogen and is essential
to platelet aggregation Platelet count normal, but bleeding
prolonged Fails to aggregate with agonists
ADPEpinephrineCollagen
May-Hegglin anomaly
Moderate macrothrombocytopenia (large platelets, low #)
Dohle-like inclusions in WBCs
Associated with abnormalities of MYH9 gene
Disorders of Pregnancy Incidental thrombocytopenia of pregnancy
Occurs in 5-7% of pregnanciesPoses no risk for neonatal thrombocytopenia
Thrombocytopenia from pre-eclampsiaPre-eclampsia is a hypertensive disorder of
pregnancy (7-10%)10-20% of pre-eclamptic women will develop
thrombocytopeniaProlonged bleedingPathogenesis unknown
Idiopathic Thrombocytopenic Purpura (ITP)--autoimmune
ACUTE CHRONIC
Children No gender preference Platelet count <20x109
Abrupt onset of bleeding Infection 1-3 wks prior Lasts 2-6 weeks
Adults Females 3:1 Platelet count 30-80x109
Insidious onset of bleeding No history of infection Lasts months to years
Drug induced platelet dysfunction Aspirin inhibits prostaglandin production and
Thromboxane A2 (TXA2) Aspirin irreversibly acetylates the cyclooxygenase
enzymes which prevents formation and release of thromboxane A2
Platelets affected by aspirin continue to circulate but are no longer capable of functioning
COX-1 Thromboxane
Aspirin resistance
Evidence suggests that significant insensitivity (5% - 60%) to aspirin occurs among patients with defined coronary disease and stroke
Some individuals may have to take alternative drugs (i.e. Plavix®)Plavix (clopidigrel) affects the ADP receptor
(P2Y12) which inhibits gp IIb/IIIa receptor that binds fibrinogen
Aspirin & Clopidogrel Effects
QUESTION
Cells involved in hemostasis are:
ErythrocytesGranulocytesLymphocytesThrombocytes
QUESTION
Normal platelets have a circulating life-span of approximately:
5 days10 days20 days30 days
QUESTION
Aspirin affects platelet function by interfering with platelets’ metabolism of:
ProstaglandinsLipidsCarbohydratesNucleic acids
QUESTION
Platelet activity is affected by:
CalciumAspirinHyperglycemiaHypoglycemia
QUESTION
Thrombocytopenia is a characteristic of:
Classic von Willebrand diseaseHemophilia AGlanzmann thrombastheniaMay-Hegglin anomaly
QUESTION
Which of the following is the most common cause of an abnormality in hemostasis?
Decreased plasma fibrinogen levelDecreased Factor VIII levelDecreased Factor IX levelQuantitative abnormality of platelets
QUESTION
Which of the following is a true statement about acute idiopathic thrombocytopenic purpura (ITP)?
It is found primarily in adultsSpontaneous remission usually occurs
within several weeksWomen are more commonly affectedPeripheral destruction of platelets is
decreased
QUESTION
Which of the following is characteristic of platelet disorders?
Deep muscle hemorrhageRetroperitoneal hemorrhageMucous membrane hemorrhageSeverely prolonged clotting times
QUESTION
Thrombocytosis would be indicated by a platelet count of:
100 x 103/μL (100 x 109/L)200 x 103/μL (200 x 109/L)300 x 103/μL (300 x 109/L)600 x 103/μL (600 x 109/L)
QUESTION
vWF antigen can be found in which of the following?MyeloblastMonoblastLymphoblastMegakaryoblast
QUESTION
Alpha granules are found on the platelet in:Peripheral zoneSol gel zoneOrganelle zoneMembranes
Secondary Hemostasis
COAGULATION FACTOR INVOLVEMENT
Secondary Hemostasis
Results in the formation of a blood clot because of coagulation factors
Forms fibrin network and thrombus that stabilize the plug/clot
Stops bleeding completely Lysis (fibrinolysis) of the clot begins and
final repair of injury takes place
Thrombin acts on fibrinogen to form fibrin strands (clot)
FACTORS WERE NAMED IN THE ORDER OF DISCOVERY,
NOT IN THE ORDER IN WHICH THEY OCCUR
Secondary Hemostasis Coagulation factors are proteins (with
exception to thromboplastin and calcium) All are involved in generating insoluble
fibrin (cascade) They can be divided into 3 families based
on properties:Fibrinogen group (thrombin sensitive) –
fibrinogen, FV, FVIII, FXIIIProthrombin group (vitamin K dependent) –
Factors II, VII, IX, X, Protein C and Protein SContact family – Factor XII, Factor XI,
prekallikrein and HMWK
Vitamin K Vitamin K is inhibited
by warfarin (Coumadin®)
All factors in the prothrombin group are affected
Individuals on warfarin need to monitor vitamin K intake (hypercoag)
Factors in RED are affected by vitamin K
QUESTION
Coagulation factors affected by coumarin (warfarin) drugs are:VIII, IX and XI, II, V and VIIII, VII, IX and XII, V and VII
ANSWER
Coagulation factors affected by coumarin drugs are:VIII, IX and XI, II, V and VIIII, VII, IX and X (vitamin K dependent)II, V and VII
Mechanism of Coagulation1. Generation of Thromboplastic activity
2. Generation of thrombin
3. Conversion of fibrinogen to fibrin
Pathways for the Coagulation Cascade
Intrinsic pathway Extrinsic pathway Common pathway
Intrinsic vs. Extrinsic
IntrinsicAll factors are contained in the blood
ExtrinsicActivated by tissue thromboplastin (FIII)Thromboplastin is released from damaged cells
and tissues outside the circulating blood
Intrinsic Pathway Circulating blood contains all
components that lead to activation of Factor X
HMWK (Fitzgerald) and prekallekrein (Fletcher) activate FXII to FXIIa
Factor XII activates FXI, which in turn, activates FXI (now FXIa) in the presence of Ca++ ions
Factor IX forms a complex with FVIII (with Ca and phospholipid on platelets) which activates FX
Factor XI and XII are “contact factors” b/c their activation is initiated by contact with subendothelial basement membrane that is exposed at the time of a tissue or blood vessel injury
Intrinsic Pathway
Also these complex reactions take place slowly, they account for the most coag activities in the body
The APTT test can monitor the intrinsic pathway
The APTT measures:
Factors XII, XI, X, IX, VIII, V, II, and fibrinogen
Common Pathway Factors
Extrinsic Pathway Occurs when tissue
thromboplastin (not in blood) enters vasculature
Factor VII is activated to VIIa by Ca++ and tissue thromboplastin (FIII)
Factor VIIa can now activate FX (common)
Extrinsic pathwayvascular injury
tissue thromboplastin + VIIa
VII
V
X
Ca++
X Xa
II (prothrombin) IIa (thrombin)
I (fibrinogen) fibrin
(VVa)
Extrinsic Pathway The extrinsic pathway can also quickly provide
small amounts of thrombin (leads to fibrin formation)
Thrombin can also enhance the activity of Factor V and VIII (intrinsic pathway)
The PT test (prothrombin time) monitors the extrinsic pathway
The PT measures Factors VII, X, V, II, and I
Common
QUESTION
Which of the following factors is used only in the extrinsic coagulation pathway?IIVVIIVIII
ANSWER
Which of the following factors is used only in the extrinsic coagulation pathway?IIVVIIVIII
Common Pathway Begins with FX Activation of FX is the point
where both the intrinsic and extrinsic pathway converge
Once Xa is formed, it’s cofactor (FV), in the presence of Ca++ and PF3 convert prothrombin to thrombin
Thrombin acts to convert fibrinogen to fibrin
Factor XII stabilizes the clot
Interpreting abnormal tests
Disorders of the coag cascade
Hemophilia AFactor VIII deficiency (VIII:C subunit)Have normal VIII:vWF moleculevWD is deficient of VIII:vWF
Hemophilia Bfactor IX deficiencyMild, moderate, or severe (need to test)
Hemophilia CFactor XI deficiency4th most common inherited bleeding disorder
Factor XII deficiency
Causes prolonged APTT Normal PT, PFA 100 and TT No signs of a bleeding disorder FXII may be more involved with
inflammation since FXII deficient individuals have higher rates of infection.
Disorders of the coag cascade
Vitamin K deficiencydiet, malabsorption disorders, liver disease
pregnancyCan prolong factor assays that include II, VII, IX,
and X…as well as the PT and APTT Found in leafy green vegetables
○ Green tea (712 μg)○ Avocado (634)○ Turnip greens (408)○ Brussels sprouts (317)○ Broccoli (200)
Direct thrombin inhibitors:
- Hirudin (leaches)
- Argatroban Vitamin K antagonists:
-Coumadin® (warfarin)
Heparins:
-Heparin
-Lovenox® (enoxaparin), LMWH
Factor Xa inhibitors:
-Arixtra®
Anti-coags that can affect the cascade
PTaPPT
Heparin Heparin and its low molecular weight
derivatives are effective at preventing deep vein thromboses and pulmonary emboli in patients at risk
Heparin binds to the enzyme inhibitor ATIII causing a conformational change that results in its activation through an increase in the flexibility of its reactive site loop
The activated ATIII then inactivates thrombin and other proteases involved in blood clotting, most notably FXa
Fibrinolysis….almost done Fibrinolysis is the last stage of coagulation
that causes the dissolution of the fibrin clot Fibrinolysis is mediated by the conversion
of plasminogen to plasmin Activation of plasminogen can be due to:
Intrinsic activation- initiated by FXIIa & kallikrienExtrinsic activation- stimuli like vascular injury,
ischemia, exercise and stressExogenous (therapeutic) activation- drugs like
streptokinase, urokinase, tissue plasminogen activator (tPA)…for strokes
ED D
FIBRINOGEN
thrombin Fibrin polymer
Factor XIII
Crosslinked fibrin polymer
“Stabilized Clot”
Fibrin monomer
plasminogen plasmin
plasmin
Degradation products
FIBRIN
D-dimer (cross-linked D-domains)
D fragments
E fragments
X fraction:
Y fraction
D fragments
Fibrinolysis testing
Most commercial FDP kits detect D & E fragments which are both products of fibrin and fibrinogen degradation (latex kits)
The D-Dimer test is a specific marker for plasmin degradation of fibrin (latex kit or ELFA)
Latex particles are coated with anti-human fibrinogen
SPR – Solid Phase Reaction
Sample goes here
Fibrinolysis
Disease states can either increase or decrease fibrinolytic activityDisseminated Intravascular CoagulationTrauma from surgical procedures or
accidentsDeficiencies in or consumption of the
various inhibitors and activators of the fibrinolytic system
DIC Uncontrolled formation of thrombi/fibrin Fibrinolysis is activated but rapidly
consumed thus leading to depletion of coag factors and platelets
Bleeding, shock, microscopic thrombiPetechiae, hematomas, deep tissue bleeding
Associated with obstetric emergencies, septicemia, burns, crush injuries, cardiac and vascular disorders
DIC lab findings
PT and PTT usually prolonged, not always
Fibrinogen – 2-3X normal (150-400) Platelets – thrombocytopenia (<50,000) FDPs – usually elevated D-Dimer – elevated
DVT Occurs when a blood
clot forms in one of the large veins, usually in the lower limbs
The blood clot can break loose and move into the:Lungs (Pulmonary
Embolism)Brain (Stroke)
DVT facts DVT occurs in about 2 million Americans every
year. Up to 600,000 people are hospitalized in the U.S.
each year for DVT. Fatal PE may be the most common preventable
cause of hospital death in the United States. In the elderly, DVT is associated with a 21%
mortality rate, and PE is associated with a 39% mortality rate.
PE is the leading cause of maternal death associated with childbirth. A woman's risk of developing emboli is six times greater when she is pregnant
Lab tests
D-Dimer is most useful b/c it tests for the breakdown of the fibrin mesh>10,000 critical (>500 pos)
FDPs are sometimes useful in post-op deep vein thrombosis
Natural anticoagulants In vivo anticoagulants help to prevent
thrombosisAntithrombin III (ATIII)
○ Inhibits thrombin and factor Xa, but can also affect Factors IXa, XIa, and XIIa
○ Deficiencies can be inherited or acquired○ ATIII can be given as treatment for thrombotic disorders
Heparin Cofactor○ ATIII heparin cofactor and HC-II are thrombin inhibitors
that are present in human plasma○ They are produced by mast cells○ Unfractioned heparin binds to ATIII to target factors IX,
X, V, and II (prothrombin)..detected by APTT or both (if dose is high
Natural Anticoagulants (cont’d)
Protein C and Protein SProtein C is made in the liver and circulates
as zymogenIt is converted to activated protein C (APC)
by thrombin and thrombomodulinAPC with its cofactor, protein S, can
inactivate FV and VIII
Protein C/S Disorders APC Resistance
Also factor V LeidenFactor V Leiden is a genetic mutation that
alters Factor V so that APC cannot bind and inactivate it
This can lead to thrombophilia (hypercoagulability…..thrombosis)
20-50% of inherited thrombophiliasDiscovered at the Univ. of Leiden
(Netherlands)
QUESTION
A deficiency of protein C is associated with which of the following?Prolonged APTTDecreased fibrinogen (<100)Increased risk of thrombosisSpontaneous hemorrhage
ANSWER
A deficiency of protein C is associated with which of the following?Prolonged APTTDecreased fibrinogen (<100)Increased risk of thrombosisSpontaneous hemorrhage
QUESTION
Which of the following is a characteristic of Factor XII deficiency?
Negative bleeding historyNormal clotting timesDecreased risk of thrombosisEpistaxis
The Coagulation Lab
A coagulation lab performs: Routine Testing
PT(INR) aPTT Fibrinogen D-Dimer FDP Thrombin Time PFA-100 Bleeding Time
Special Coag Factor Assays Lupus Panels Mixing Studies ATIII Plasminogen von Willebrand LMWH Euglobulin clot lysis Factor XIII Factor Inhibitor Fletcher Factor APC Resistance Antiphospholipid testing (ELISA) HIT (ELISA) PAI-1 (ELISA)
Coagulation Basics 3.2% sodium citrate tubes
Anticoag to blood ratio is cruciala short draw could prolong results
Ratio is 9 parts whole blood to 1 part anticoagulant (sodium citrate)
Discard first tube b/c it may be contaminated with tissue fluid (tissue thromboplastin may activate extrinsic system..prolonged PT)
Anticoagulants to remember
Anticoagulants (EDTA, citrate) remove calcium to prevent clotting (in vitro)
Heparin (FIX, X, V, II) and warfarin (vitamin K dependent factors II, VII, IX, X) prevent the conversion of prothrombin (factor II) to thrombin (in vivo)
Centrifugation Samples are spun in a refrigerated centrifuge fro
10-15 minutes at 3500 rpms to obtain platelet poor plasmaRefrigeration maintains labile factors VIII and V
Samples for assays that are performed at a later date are:Immediately separatedChecked for platelet count (<10x109)Phospholipids from lysed platelets can falsely effect
phospholipid based tests (Lupus testing)frozen rapidly at -72°CThawed rapidly to avoid cryoprecipitation (VIII)
Automated Testing Automated (photooptical) or semi-automated End point of reaction is a clot (measured in
seconds) Clot formation is timed automatically and is
detected by a photocell that reads the optical density change when the clot is formed
Contains a heat block to bring reagents and plasma to 37°C Reagents not in use are kept at 4-6°C to maintain integrity
(on instrument, in fridge, or cooling block) Analyzers automatically pipette (or require manual
pipetting) PT, APTT, factor assays, fibrinogen, etc
STart 4 Analyzer
Electromechanical method
PT testing Prothrombin time (extrinsic & common
pathways) Method of choice for monitoring Coumadin
(Vit K antagonist) Reagents include
Calcium ChlorideThromboplastinBoth of these are needed to initiate the extrinsic
pathway and test for any deficiencies
Contained together in a
single vial
PT Testing
Normal PT = 10-14 seconds In the past, many labs performed a
PT test with (thromboplastin) and a separate PT(INR) test for those on oral anticoagulant therapy to determine an INR
Now, some labs run the PT(INR) on all patients and have discontinued the routine PT
PT INR INR – International PT standardization for
oral anticoagulant therapy monitoring The WHO and the International Committee
on Thrombosis and Hemostasis recommend that PT tests run on patients on oral therapy use an INR value
INR values are independent of the reagents and methods used, and are specifically intended for assessing patients stabilized on long term oral anticoagulant therapy
PT(INR)
To standardize the PT, a calculation is used to obtain the International Normalized Ratio (INR) for people on warfarin (normal INR = up to about 4)
INR =Patient PT
Mean PT (of normal population)X ISI
International Sensitivity Index
Current normal PT in lab = 9.4-12.5
PT INR Note:
When coumadin is initiated, with a loading dose, there is a rapid depletion of Factor VII during the first 48 hrs, with other factors not being fully affected until about 5 days after
Prolonged PT times before 5 days have no correlation with any therapeutic benefits
Measurements are not suggested until the 5th day of therapy
QUESTION
The prothrombin time (PT) test requires that the patient’s citrated plasma be combined with:Platelet lipidsThromboplastinCa++ and platelet lipidsCa++ and thromboplastin
ANSWER
The prothrombin time (PT) test requires that the patient’s citrated plasma be combined with:Platelet lipidsThromboplastinCa++ and platelet lipidsCa++ and thromboplastin
APTT Testing Activated Partial Thromboplastin Time Screens for deficiencies involving factors of
the intrinsic pathwayMost sensitive to Factors VIII, IX, X, XI & XIICan be prolonged in severe Factor V deficiency
Fresh citrated plasma provides all factors necessary for the intrinsic clotting mechanism except ionic Ca (removed by citrate)platelet factor (removed by centrifugation
APTT testing An activator and phospholipid is incubated with
patient plasma to activate contact factors (creates negatively charged surface) Activators: kaolin, celite, ellagic acid, silica Phospholipids: rabbit brain, cephalin, soy bean
Calcium is added after incubation and the time it takes for a clot to form observed
Normal aPTT = varies from about 24-34 seconds Actin® is a reagent that contains ellagic acid and
rabbit brain
APTT
Note:Heparin can be given continuously (IV) or in
subcutaneous mini-dosesCare should be taken not to obtain samples
above heparin linePatients should be monitored prior to next
dose if APTT is to be meaningfulHeparin contamination can sometimes
severely prolong APTT (>60 seconds)
Another APTT reagent: PSL Panthromtin SL is more sensitive than
the routine APTT reagentIt is used in some labs to detect inhibitors
affecting the APTTThe “SL” refers to Lupus and is used to
detect risk of diseaseIf prolonged and heparin is ruled out, further
Lupus testing is performed
Fibrinogen Testing Thrombin converts fibrinogen to fibrin Thrombin is added to plasma and the time
it takes to clot (determined in seconds) is proportional to the fibrinogen concentration in plasma
Calibration curves are needed for each new lot of thrombin
Normal range = 150 – 400 mg/dL Anything >500 is run manually on a semi-
automated instrument (i.e. Start 4)
SPECIAL COAG TESTING
Factor Assays Allows testing for specific factors by
making a series of dilutions on a control, reference (deficient) plasma, and patient
3 (automated) dilutions made: 1:10, 1:20, 1:40
Run a PT or APTT, depending on what factor is being tested
The factor deficient plasma has every factor except the desired factor
Factor assays PT: 2, 7, 5, 10 PTT: 9, 8, 11, 12 The 3 points of the reference (calibration curve) is
plotted and a best fit line is drawn (automated) The 3 points of the patient or control are plotted
and compared to referenceIf patient is below reference, then the factor activity is
low (normal 50-150%)If patient is above, then factor is high (no worry)If patient crosses the reference line (not parallel), then
an inhibitor is suspected
Factor graph (basic example)
Curvilinear paper is used to obtain straight line
1:10
1:40
1:20
reference
Low factorInhibitor present, possibly FVIII inhibitor or
Lupus anticoagulant
Factor XIII Stabilizes clot Uses 2 tubes containing plasma are clotted
with Calcium chloride5M urea added to one tubeAcetic acid added to the other
Checked (in water bath) at 30 min intervals to monitor clotIf clot dissolves in 10-30 min, FXIII deficiencyNormal clots remain over an hourUmbilical stump hemorrhage is the hallmark of
FXIII deficiency
Lupus Anticoagulant Panel LA (or inhibitor) is thought to be a
nonspecific phospholipid antibody Prolonged PTT is a routine screening
test If after testing, a lupus inhibitor is
detected, mixing studies are performed to rule out factor deficiency
LA testing Lupus panel consists of:
APTT: Actin FSL and Pathromtin SL Dilute Russell’s Viper Venom Test (DRVVT) – modified
APTT that uses viper venom as an activator. Very sens. DRVV Confirm – higher concentrations of phospholipids.
This higher concentration of phospholipids allows the reagent to “correct” the prolonged DRVVT.
Anti-cardiolipin, anti-phosphatidylserine, anti-prothrombin and beta-2-glycoprotein
Sta-Clot kit detects lupus inhibitor○ Tube A is an APTT○ Tube B is an APTT with phospholipid○ If clotting changes more than 8 seconds, it is positive
Triturus tests (ELISA)
Antiphospholipid tests (for APS syndrome, SLE, etc)
HIT Testing PAI-1 Inhibitor
HIT Heparin Induced Thrombocytopenia
Tests for anti-platelet antibodies to the PF4 receptor on platelets
In patients on heparin or who have had previous exposure to heparin
Platelet count dramatically drops by 50% after second exposure
Must be removed from heparin and given alternative treatment (i.e. argatroban, LMWH)
Euglobulin Lysis Overall screen of the fibrinolytic system Fibrinogen, plasmin and fibrinolytic agents are
precipitated (left in tube) Inhibitors of fibrinolysis are discarded The precipitate is re-dissolved in buffer and
thrombin is added to form a clot It is put in a water bath and monitored every 10
min for 1 hour then at 2 and 24 hoursNormal patient >2 <24 hoursAbnormal <2 hrs increased fibrinolytic activity>24 hours decreased fibrinolytic activity
ATIII testing
Naturally occurring inhibitor that neutralizes the serine protease thrombin, FIXa, Xa, Xia and XIIa
The inhibition of thrombin by antithrombin is greatly accelerated by heparin
Chromogenic assays that inhibit thrombin or Fxa, microlatex assays, nephalometry are frequent assays.
Bleeding Time Tests for platelet function Normal 3-9 minutes Patient MUST have a Plt count >50,000 Blood pressure cuff is applied (40) Small incision made with lancet and the bleeding is
“wicked” with paper disc every 30 seconds until plug forms
Test is terminated if bleeding does not stop after 15 minutes
Abnormal bleeding disorders include: Thrombasthenia vonWillebrand disease Bernard Soulier Aspirin sensitivity
PFA 100®
Platelet Function Analyzer Simulates in vivo conditions Uses 2 cartridges:
Col/EPI Col/ADP
Could replace the bleeding time Francis J., Francis, D., Larson, L., Helms, E. & Garcia, M.
(1999). Can the Platelet Function Analyzer (PFA)-100 test substitute for the template bleeding time in routine clinical practice? Platlets, 10(2-3), 132-6.
Assesses platelet dysfunction due to: ASA-like platelet defects congenital platelet defects
(vWD)
PFA 100
800 µL blood
Filter with Epinephrine or ADP
Platelet aggregation/plug
Once started, a timer begins. When platelets have plugged aperature, the final
time is reported as “closure time”
Interpretation
Col/EPI Col/ADP
True platelet defect (vWD, etc)
+++(ex. >300)
+++
Aspirin-like defect (ASA)
+++ normal
Platelet Aggregation
A photo optical instrument or instruments using electrical currents are used to detect aggregation of platelets in platelet rich plasma or whole blood, respectively
PRP or whole blood is mixed with an aggregating agent and results are transmitted to a chart recorderCollagen, ADP, epinephrine, thrombin,
serotonin, ristocetin
Platelet aggregation curves
http://www.practical-haemostasis.com/Data%20Interpretation/Data%20Questions/data_interpretation_platelet_function.html click here for practice cases
Aggregation Disorders
Lab Findings Glanzmann’s Thrombasthenia
Bernard-SoulierSyndrome
Platelet Aggregation
ADP Abnormal Normal
Thrombin Abnormal Abnormal
Collagen Abnormal Normal
Epinephrine Abnormal Normal
Ristocetin Normal Abnormal
vWF Normal Abnormal
QUESTION
Heparin Induced Thrombocytopenia (HIT) is an immune mediated complication associated with heparin therapy. Antibodies are produced against:ACLAPF4ATB2GP1
QUESTION
A bleeding time is used to evaluate the activity of:
PlateletsProthrombinLabile factorFactor XIII
QUESTION
A patient has been taking aspirin regularly for arthritic pain. Which one of the following tests is most likely to be abnormal in this patient?Platelet countTemplate bleeding timeProthrombin timeActivated partial thromboplastin time
QUESTION
Biological assays for antithrombin III (ATIII) are based on the inhibition of:
Factor VIIIHeparinSerine proteasesAnti-ATIII globulin