renee wilkins, ph.d., mls(ascp) cm mls 322 clinical hematology school of health related professions...

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