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Antiplatelet Medication Response Testing: Aspirin and
Clopidogrel
Leah Coppolino, MPH, MLS(ASCP), CLS(NCA)
Reviewers: Michael P. Ero, MLS(ASCP), CLS(CA), MBA; Dr. Phyllis Ingham EdD. MEd. MT(ASCP)
Course Instructions

Please proceed through the course by clicking on the blue arrows or text links. Use the table of contents to monitor your progress. Your progress will be
saved automatically as you proceed through the course, and you may later continue where you left off even if you use a different computer. You may
encounter practice questions within the course, which are not graded or recorded.
Course Info

This course carries the following continuing education credits:

Florida Board of Clinical Laboratory Science CE ‐ General ﴾Hematology﴿: 1.00 hour﴾s﴿

Course Number: 909566
P.A.C.E. Contact Hours: 1.00 hour﴾s﴿
Course Number: 578‐031‐22

Level of instruction: Intermediate

Intended Audience: Medical laboratory scientists, medical technologists, and technicians. This course is also appropriate for medical laboratory science
students and pathology residents.

Author information: Leah Coppolino, MPH, MLS﴾ASCP﴿CM is a Program Director at MediaLab, Inc. Previously, she was the Director of the Medical
Laboratory Science Program at St. Christopher’s Hospital for Children in Philadelphia, Pennsylvania. She holds a Masters in Public Health from Thomas
Jefferson University.

Reviewer information: Michael P. Ero, MLS﴾ASCP﴿, CLS﴾CA﴿, MBA is the Founder and President of Machaon Diagnostics, a clinical reference laboratory
specializing in bleeding and clotting disorders. Michael has over three dozen publications in the field of thrombosis and hemostasis and has presented his
work at national and international scientific meetings. He has been primary investigator on over 130 clinical trials and studies with physical, molecular,
immunologic or hemostatic endpoints. He is currently an adjunct instructor at the School of Medical Technology at Michigan State University. He was
previously the vice president of the Coagulation Center, Inc. of Oakland, California overseeing the delivery of comprehensive coagulation and platelet
services. He has held past clinical‐ and research‐focused positions with The Scripps Research Institute, Loyola University Medical Center and the
University of Illinois, Chicago. He holds an MBA from Babson College in Wellesley, Massachusetts and a BS in Medical Technology from Michigan State
University.

Reviewer information: Dr. Phyllis Ingham is the Director of Clinical Laboratory Technology Phlebotomy at West Georgia Technical College in Waco
Georgia. Dr. Ingham holds a BS degree from Auburn University, a masters degree in education from Alabama State University, and a Doctorate in
Educational Leadership from Argosy University. Dr. Ingham serves as District V Director for ASCLS‐GA.
 Course Introduction
Introduction

Antiplatelet medications are used in the treatment and prevention of adverse thrombotic and ischemic events. Two important antiplatelet medications that
are commonly used are aspirin, a cyclooxygenase inhibitor, and clopidogrel, an ADP receptor inhibitor. This course will discuss the mechanism of action
and the limitations for each drug.
Platelet function assays may be ordered to evaluate patient response to antiplatelet medications. Patient monitoring is important because of bleeding risks
posed by use of these medications and the risk for future cardiac events, if testing indicates a lack of response to the medication. A variety of methods for
assessing platelet function are available. Some of the more widely used procedures will be covered in this course.

Aspirin Therapy and Introduction

Platelets and Aspirin to Aspirin Resistance

Blood clots are formed through the process of hemostasis. In primary

hemostasis, platelets activate and aggregate to form a platelet plug. This
platelet plug is then strengthened by fibrin and a thrombus is formed
during the process of secondary hemostasis. Hemostasis is essential to
prevent excessive loss of blood. However, when a thrombus forms inside
an artery, it blocks the flow of blood to that tissue. This causes a
myocardial infarction, if a coronary artery supplying blood to the muscle
of the heart is blocked, or a stroke, if a thrombus forms in an artery
supplying blood to the brain. In high‐risk patients, it is critical to prevent
the formation of thrombi in these arteries. This is where aspirin becomes
helpful.
Aspirin prevents thrombi formation by blocking platelet production of
thromboxane A2, an essential platelet aggregation component. Aspirin has
the ability to suppress the creation of prostaglandins and thromboxane A2
by irreversibly inactivating the cyclooxygenase‐1 ﴾COX‐1﴿ enzyme. COX‐1
plays an important role in prostaglandin and thromboxane A2 synthesis.
Although other nonsteroidal anti‐inflammatory drugs ﴾NSAIDs﴿ can also
inhibit the COX‐1 enzyme in a reversible manner, aspirin inhibits the COX‐
1 enzyme for a greater period of time, thus making it the preferred NSAID
for the prevention of ischemic events.

Aspirin Therapy and Introduction

Aspirin Dosing to Aspirin Resistance

Daily low doses of aspirin are most commonly prescribed to patients with a high risk of thrombi production to produce an antiplatelet effect. When
aspirin is given in low doses ﴾75‐150 mg/day﴿, the maximal antiplatelet effect may take several days. At a dose of 160‐325 mg/day, the maximal
antiplatelet effect of aspirin is faster and can occur in approximately 30 minutes. Therefore, aspirin at low doses is prescribed for the long‐term prevention
of heart attacks and strokes and moderate doses are given in situations where an immediate anti‐clotting effect is needed.

Low‐Dose ﴾~75‐150 mg/day﴿ Recommendations:

Patients undergoing surgery to open or bypass blocked arteries
Patients who have previously had heart attacks or strokes
Patients with risk factors, such as diabetes
Patients with peripheral vascular disease

Moderate‐Dose ﴾~160‐325 mg/day﴿ Recommendations:

During a myocardial infarction
Patients who are experiencing unstable angina
Patients who are experiencing ischemic strokes

Aspirin Therapy and Introduction

Aspirin Resistance to Aspirin Resistance

Several studies conducted within the past five years concluded that 5‐40% of individuals who take aspirin every day to help prevent heart attacks and
strokes may not benefit from the anticoagulant effect of aspirin.* A person is considered aspirin‐resistant if:
The individual is using aspirin therapeutically ﴾75‐150 mg/day﴿ and an ischemic cardiac event occurs
Laboratory testing determines a lack of antiplatelet effect, despite therapeutic dosing for at least five days

An algorithm, such as the one shown on the following page, may be a useful tool for determining whether platelet function testing should be ordered.

* It should be noted that a study published in 2012 concluded that aspirin resistance is very rare. However, the sample group of 400 individuals in this
strokes may not benefit from the anticoagulant effect of aspirin.* A person is considered aspirin‐resistant if:
The individual is using aspirin therapeutically ﴾75‐150 mg/day﴿ and an ischemic cardiac event occurs
Laboratory testing determines a lack of antiplatelet effect, despite therapeutic dosing for at least five days

An algorithm, such as the one shown on the following page, may be a useful tool for determining whether platelet function testing should be ordered.

* It should be noted that a study published in 2012 concluded that aspirin resistance is very rare. However, the sample group of 400 individuals in this
study were all relatively young and healthy. In previous studies, sample groups included individuals who were at high risk for cardiac events, for example,
a cohort of patients with metabolic syndrome. An abstract of the article for the 2012 study is available at:
http://circ.ahajournals.org/content/early/2012/12/04/CIRCULATIONAHA.112.117283. Accessed April 20, 2020.

Aspirin Therapy and Introduction

Algorithmic Approach For a Patient With Suspected Aspirin
to Aspirin Resistance
Resistance

* PVD: peripheral vascular disease

Reference: Mansour K, Taher AT, Musallam KM, Alam S. Aspirin Resistance. Advances in Hematology, vol. 2009, Article ID 937352, 10 pages, 2009.
doi:10.1155/2009/937352.

Aspirin Therapy and Introduction

Factors that May Limit the Effectiveness of Aspirin
to Aspirin Resistance

Various factors may limit the effectiveness of aspirin, including:

Use of enteric coated aspirin
Buildup of plaque in blood vessels, causing blood vessel constriction
despite the use of aspirin
Other medications that prevent aspirin from binding to
cyclooxygenase receptors, including nonsteroidal anti‐inflammatory
drugs ﴾NSAIDs﴿, especially ibuprofen
Other blood‐clotting processes or other platelet activation pathways
that exert a stronger effect than the antiplatelet effect of aspirin
Increased platelet turnover
Cyclooxygenase enzyme genetic mutations
 cyclooxygenase receptors, including nonsteroidal anti‐inflammatory
drugs ﴾NSAIDs﴿, especially ibuprofen
Other blood‐clotting processes or other platelet activation pathways
that exert a stronger effect than the antiplatelet effect of aspirin
Increased platelet turnover
Cyclooxygenase enzyme genetic mutations

Aspirin Therapy and Introduction

Ungraded Practice Question
to Aspirin Resistance

True or False: Aspirin's ability to suppress prostaglandins and thromboxane A2 creation is due to its irreversible inactivation of the cyclooxygenase ﴾COX‐1﴿
enzyme.

Select true or false

True

False

Aspirin Therapy and Introduction

Ungraded Practice Question to Aspirin Resistance

True or False: Aspirin's ability to suppress prostaglandins and thromboxane A2 creation is due to its irreversible inactivation of the cyclooxygenase ﴾COX‐1﴿
enzyme.

Select true or false

True

False

Feedback

This statement is true. Aspirin prevents thrombi formation by blocking the production by platelets of thromboxane A2, an essential platelet aggregation
component. Aspirin has the ability to suppress prostaglandins and thromboxane A2 creation by inactivating the COX‐1 enzyme.

Clopidogrel Therapy and

Platelets and Clopidogrel
Clopidogrel Resistance

Clopidogrel functions by irreversibly inhibiting a receptor called P2Y12,

an adenosine diphosphate ﴾ADP﴿ receptor located on platelet cell
membranes. This receptor is important in the process of platelet activation
and of fibrin cross‐linking in secondary hemostasis.
The blockage of this receptor inhibits platelet aggregation by blocking
activation of the glycoprotein IIb/IIIa pathway. The IIb/IIIa complex
functions as a receptor, mainly for fibrinogen, in the hemostatic process.
Ultimately, the fibrin cross‐linking is inhibited, leading to the desired
antithrombotic ﴾anticoagulant﴿ activity of the medication.

Clopidogrel Therapy and

Clopidogrel Dosing
Clopidogrel Resistance

Clopidogrel is used in the treatment of various cardiovascular conditions. These conditions have their own recommended dosages as shown below.
Platelet inhibition can often be demonstrated two hours after a single dose of oral clopidogrel. However, the absorption and distribution of the drug may
be too slow for some conditions. Therefore, a loading‐dose of 300 mg may need to be administered. The table below lists dosage recommendations for
various conditions. In all cases, clopidogrel is given orally.
Clopidogrel Dosing
Clopidogrel Resistance

Clopidogrel is used in the treatment of various cardiovascular conditions. These conditions have their own recommended dosages as shown below.
Platelet inhibition can often be demonstrated two hours after a single dose of oral clopidogrel. However, the absorption and distribution of the drug may
be too slow for some conditions. Therefore, a loading‐dose of 300 mg may need to be administered. The table below lists dosage recommendations for
various conditions. In all cases, clopidogrel is given orally.

Condition Recommended
Dosage
Recent myocardial infarction ﴾MI﴿, recent stroke, or peripheral 75 mg once daily
arterial disease ﴾PAD﴿
Coronary artery disease ﴾CAD﴿ 75 mg once daily
Secondary prevention of cardioembolic stroke ﴾patient not a 75 mg once daily
candidate for oral anticoagulation, eg, warfarin﴿
Acute coronary syndrome ﴾ACS﴿ 300 mg initially followed
by 75 mg daily

Clopidogrel Therapy and

Clopidogrel Resistance
Clopidogrel Resistance

Since antiplatelet therapy has become a key component in cardiovascular and neurovascular medicine, it is important to monitor the effectiveness of
these medications. Recent studies have demonstrated an emergence of the new clinical entity of "clopidogrel resistance," also referred to as clopidogrel
nonresponsiveness. Clopidogrel is marketed under the trade name, Plavix®. Studies have shown that as many as 30% of patients do not respond
adequately to standard doses of clopidogrel ﴾similar to the statistics in aspirin resistance﴿.
There are several possible mechanisms of clopidogrel resistance, including:
Genetic variants that alter or inhibit appropriate clopidogrel metabolism
Noncompliance in taking clopidogrel as prescribed
Interference from other medications
Accelerated platelet turnover, with introduction into bloodstream of newly formed, drug‐unaffected platelets that can negatively influence
antiplatelet effect of clopidogrel

Age, diabetes mellitus, decreased left ventricular function, renal failure, and acute coronary syndrome are all associated with decreased clopidogrel
function.

Clopidogrel Therapy and

Ungraded Practice Question
Clopidogrel Resistance

Which of the following is a receptor that is located on platelet cell membranes and is inhibited by clopidogrel, thereby preventing platelet aggregation?

Please select the single best answer

vWF

Aspirin

CYP2D6

P2Y12

Clopidogrel Therapy and

Ungraded Practice Question
Clopidogrel Resistance

Which of the following is a receptor that is located on platelet cell membranes and is inhibited by clopidogrel, thereby preventing platelet aggregation?

Please select the single best answer

vWF

Aspirin

CYP2D6

P2Y12
 Aspirin

CYP2D6

P2Y12

Feedback

The correct answer if P2Y12. This is an adenosine diphosphate ﴾ADP﴿ receptor located on platelet cell membranes. This receptor is important in the
processes of platelet activation and fibrin cross‐linking in secondary hemostasis. When this receptor is blocked by clopidogrel, the formation of thrombi is
inhibited. Therefore, this treatment is used commonly for those at high risk of ischemic cardiovascular events.

Antiplatelet Medication Response

Specimen Collection for Platelet Function Testing
Testing: Specimen Collection and
Handling Requirements

Most instruments used for platelet function testing require the use of
citrated blood. Blood is collected by venipuncture into a plastic tube
containing buffered 3.2% sodium citrate, which is then mixed gently
immediately upon filling.
Collection tubes must be filled completely to ensure the proper 9:1 ratio
of blood to anticoagulant. Alterations in the blood to anticoagulant ratio
can have an adverse effect on platelet response to agonists ﴾platelet
activators﴿ used in the test procedure. It is also important to the outcome
of the test that the venipuncture is not traumatic as this can result in
platelet activation. A needle gauge size between 19 and 21 is
recommended to prevent vein trauma or reduced blood flow, leading to
activated platelets. If the vein collapses or there is stoppage of blood flow
during collection, the specimen should be discarded and a venipuncture
should be repeated from another site.
Some test manufacturers may recommend that the whole blood
specimen rest on the laboratory bench top for 30 minutes prior to platelet
function testing. Platelets, which may be activated as a result of
venipuncture, will return to an inactive ﴾resting﴿ state during the 30
minutes. Specimens should be held at room temperature ﴾18‐24°C﴿ only
and not refrigerated or frozen. Testing should be completed within the
time limit specified by the manufacturer to ensure platelet viability
﴾usually within 3‐4 hours of collection﴿; platelet functionality becomes
abnormal after a period of approximately 3‐4 hours.
Specimens for platelet function testing should be rejected if they are
clotted, grossly hemolyzed, or if the collection time limit has been
exceeded.

Antiplatelet Medication Response

Specimen Processing for Platelet‐Rich Plasma
Testing: Specimen Collection and
Handling Requirements

Platelet‐rich plasma ﴾PRP﴿ may be required for some platelet analysis

procedures, such as optical light transmission aggregometry. PRP is
prepared by centrifuging whole blood at a low relative centrifugal force ﴾g
force﴿. These procedural steps should be followed:
1. Collect an anticoagulated sample using a tube containing 3.2%
sodium citrate; ensure proper filling of the tube.
2. Prepare the plasma specimen by centrifuging at a low g force ﴾eg,
170 g for 15 minutes﴿.
3. Ensure that interfering substances are not present in the sample
﴾lipemia, hemolysis, etc.﴿.
4. Analyze the PRP sample within the time limit recommended by the
instrument manufacturer ﴾usually within 3‐4 hours of collection﴿.
5. Ideal concentrations of platelet in platelet‐rich plasma are 200‐300 x
Hover over the image to zoom in.
109/L. Platelet‐poor plasma may be used to dilute higher
concentrations of platelet‐rich plasma, in some cases.

To obtain accurate results during analyses utilizing platelet‐rich plasma,

samples with baseline platelet counts above 100 x 109/L are preferred.

Antiplatelet Medication Response

Ungraded Practice Question
To obtain accurate results during analyses utilizing platelet‐rich plasma,
samples with baseline platelet counts above 100 x 109/L are preferred.

Antiplatelet Medication Response

Ungraded Practice Question Testing: Specimen Collection and
Handling Requirements

What is the recommended platelet count for a platelet‐rich plasma sample, such as those prepared for light transmission aggregometry?

Please select the single best answer

600 ‐ 800 x 109/L

200 ‐ 300 x 109/L

50 ‐ 150 x 109/L

Antiplatelet Medication Response

Ungraded Practice Question
Testing: Specimen Collection and
Handling Requirements

What is the recommended platelet count for a platelet‐rich plasma sample, such as those prepared for light transmission aggregometry?

Please select the single best answer

600 ‐ 800 x 109/L

200 ‐ 300 x 109/L

50 ‐ 150 x 109/L

Feedback

The correct answer is 200 ‐ 300 x 109/L. There must be an adequate number of platelets present in the plasma to react with the agonists in the testing
system to produce a thrombus, therefore, sample containing 50‐150 x 109/L platelets would not be adequate. In addition, platelet‐rich plasma with 600‐
800 x 109/L platelets, may not produce accurate results during evaluations such as optical light transmission.

Aspirin and Clopidogrel Response

Monitoring Response to Aspirin and Clopidogrel Therapy
Testing

Laboratory analysis of response to aspirin and/or clopidogrel therapy can play an important role in monitoring patients at high risk for adverse cardiac
events. This section of the course will discuss different analytic methodologies, some which can be utilized for both asprin resistance testing and
clopidogrel resistance testing, and others that are unique to aspirin or clopidogrel testing. Examples of the test methods which will be discussed include:
optical light transmission analysis, whole blood impedance aggregometry, Helena Laboratories PlateletWorks Kits, and cartridge‐based platelet
aggregation testing.
One of the main test methods available to analyze aspirin or clopidogrel resistance is through platelet aggregation in vitro. This method measures platelet
function and determines aspirin or clopidogrel response based on clotting time results. Agonists are used. Platelets with impaired functionality result in
increased clotting times. If the sample does not show the inhibition of platelet aggregation formation within the reference intervals defined for aspirin or
clopiogrel therapy, the individual may have a lessened response or be unresponsive to therapy. We will compare various methods that utilize this principle
in upcoming course pages.

Aspirin and Clopidogrel Response

Aspirin Response Analysis: Optical Light Transmission
Testing: Optical Light Transmission
Aggregometry

Patient platelet‐rich plasma ﴾PRP﴿ is reacted with a series of platelet‐activating reagents ﴾agonists﴿ such as arachidonic acid, collagen, or ADP, to induce
platelet aggregation. The reactions take place sequentially in an aggregometer, a spectrophotometer with a strip recorder. As platelets aggregate and a
thrombus forms, the turbidity of the platelet‐rich plasma in the cuvette declines and the amount of light transmitted to the photometer increases. These
changes are indicated by characteristic tracings on the strip recorder. Platelet dysfunction is indicated by lack of platelet aggregation.
A test result, using this method, of ≥20% aggregation in response to arachidonic acid ﴾0.5mmol/L ‐ 1.6 mmol/L﴿ and/or ≥70% aggregation in response to
10 µmol/L ADP is generally interpreted as inadequate response to aspirin therapy. This means that the patient may still be at increased risk for a
cardiovascular event.
platelet aggregation. The reactions take place sequentially in an aggregometer, a spectrophotometer with a strip recorder. As platelets aggregate and a
thrombus forms, the turbidity of the platelet‐rich plasma in the cuvette declines and the amount of light transmitted to the photometer increases. These
changes are indicated by characteristic tracings on the strip recorder. Platelet dysfunction is indicated by lack of platelet aggregation.
A test result, using this method, of ≥20% aggregation in response to arachidonic acid ﴾0.5mmol/L ‐ 1.6 mmol/L﴿ and/or ≥70% aggregation in response to
10 µmol/L ADP is generally interpreted as inadequate response to aspirin therapy. This means that the patient may still be at increased risk for a
cardiovascular event.

Aspirin and Clopidogrel Response

Clopidogrel Response Analysis: Optical Light Transmittance Testing: Optical Light Transmission
Aggregometry
In the same procedure for clopidogrel resistance testing, ADP and other agonists are used to evaluate the effectiveness of clopidogrel's blockade of the
P2Y12 ﴾ADP﴿ receptor. Significant inhibition ﴾>50%﴿ of ADP‐induced platelet aggregation, with normal platelet response to an alternative agonist, such as
thrombin receptor activating peptide ﴾TRAP﴿, indicates successful clopidogrel‐induced platelet inhibition. This assay may also be used for other P2Y12
inhibitors such as prasugrel and ticagrelor.

Aspirin and Clopidogrel Response

Aspirin Response Analysis: Whole Blood Impedance
Testing: Whole Blood Impedance
Aggregometry Aggregometry

Impedance aggregometers, such as those manufactured by Chrono‐log,

determine platelet aggregation in whole blood. This methodology does
not require the preparation of platelet‐rich plasma since whole blood is
used in the test. The instrument measures the change in resistance
﴾impedance﴿ between two electrodes immersed in the whole‐blood
sample as platelets adhere to the electrode wires and aggregate in
response to agonists added to the sample.
A two‐wire electrode is inserted in the sample cuvette containing the
patient's whole blood sample. A small voltage is applied across the Hover over the image to zoom in.
electrode, and the impedance caused by the platelets coating the wires is
measured.
Before an agonist is added, the interactions between the platelets and the
electrodes stabilize and the impedance between the two electrode wires
becomes constant, creating a baseline reading. When an agonist is added,
platelets in the specimen are activated and begin to aggregate. The
platelet coating on the immersed wires thickens over the next several
minutes, increasing the electrical impedance between the electrode wires.
This change in impedance is directly proportional to the extent of platelet
aggregation and is indicated on the digital display after a specified time.
Platelet aggregation is reported as the change in electrical impedance and
is expressed in ohms.
For aspirin response testing, arachidonic acid is used as an agonist at
concentrations between 0.5 mmol/L to 1.0 mmol/L. A decrease in platelet
aggregation for patients receiving aspirin therapy is considered a normal
response. Platelet aggregation that is not halted in the presence of
arachidonic acid may indicate a decreased response to antiplatelet
medication.
*Image adapted from Rotem.de

Aspirin and Clopidogrel Response

Clopidogrel Response Analysis: Whole Blood Impedance
Testing: Whole Blood Impedance
Aggregometry Aggregometry

Whole blood methods that detected platelet aggregation using electrical impedance technology can also be utilized for clopidogrel response testing. In
one method, ADP is used as the agonist, typically at a concentration of 5‐20 µmol/L. If a patient's test result shows a low response ﴾ADP‐induced
impedance exceeding 5 Ω ﴾ohms﴿﴿, it could indicate clopidogrel non‐responsiveness.

Aspirin and Clopidogrel Response

Ungraded Practice Question
Testing: Whole Blood Impedance
Aggregometry

True or False: Arachadonic acid is routinely used as an agonist to assess clopidogrel effectiveness in whole blood impedance aggregometry methods.

Select true or false

True
 Aggregometry

True or False: Arachadonic acid is routinely used as an agonist to assess clopidogrel effectiveness in whole blood impedance aggregometry methods.

Select true or false

True

False

Aspirin and Clopidogrel Response

Ungraded Practice Question
Testing: Whole Blood Impedance
Aggregometry

True or False: Arachadonic acid is routinely used as an agonist to assess clopidogrel effectiveness in whole blood impedance aggregometry methods.

Select true or false

True

False

Feedback

This statement is false. ADP is the agonist that is routinely used to assess clopidogrel effectiveness in whole blood impedance aggregometry methods.

Aspirin and Clopidogrel Response

Aspirin Response Analysis: Helena Laboratories
Testing: Helena Laboratories
PlateletWorks Kit PlateletWorks Kit

Plateletworks® by Helena Laboratories is actually a variety of kits used to assess patient response to different antiplatelet medications, including aspirin
and clopidogrel. For aspirin‐resistance, the kit includes arachidonic acid ﴾AA﴿ coated tubes to be utilized in an in vitro diagnostic screening test performed
on whole blood for the qualitative determination of platelet inhibition by aspirin. Aspirin inhibits AA‐induced platelet aggregation. The change in platelet
count due to activation and aggregation of functional platelets is measured using an electronic impedance‐based cell counter.
The Plateletworks methodology is an adaptation of platelet aggregometry, using a cell counter to measure total platelet count in a whole blood sample
and then to redetermine the platelet count on a second sample that has been exposed to a known platelet agonist, such as AA ﴾or ADP, if clopidogrel
response is being evaluated﴿. The agonist will stimulate those platelets that are functional to aggregate into clumps, and they will not be counted as
platelets in the second sample. The difference in the platelet count between samples one and two provides a direct measurement of platelet aggregation
and is reported as percent aggregation. Platelets rendered inactive or non‐functional by aspirin or clopidogrel do not aggregate.
The percent platelet aggregation is then calculated:
From the % Aggregation/Inhibition Chart supplied in the packaged tubes
Using the Plateletworks Calculation Wheel
Using the formula below

Baseline Platelet Count ‐ Agonist Platelet Count x 100 = % Aggregation

Baseline Platelet Count

Normal ﴾positive﴿ = >60% Aggregation

Abnormal ﴾negative﴿ = <60% Aggregation

Aspirin and Clopidogrel Response

Clopidogrel Response Analysis: Helena Laboratories Testing: Helena Laboratories
PlateletWorks Kit PlateletWorks Kit

The PlateletWorks kit that measures clopidogrel resistance utilizes ADP‐coated tubes. Clopidogrel inhibits platelet aggregation by binding to ADP
receptors, blocking this mechanism from allowing fibrinogen linking. The change in platelet count due to activation and aggregation of functional platelets
is measured using an electronic impedance‐based cell counter.
The difference in the platelet count between samples one and two provides a direct measurement of platelet aggregation and is reported as percent
aggregation. Platelets rendered inactive or non‐functional by clopidogrel do not aggregate.
The percent platelet aggregation is then calculated:
From the % Aggregation/Inhibition Chart supplied in the packaged tubes
Using the Plateletworks calculation wheel
Using the formula below

Baseline Platelet Count ‐ Agonist Platelet Count x 100 = % Aggregation

The percent platelet aggregation is then calculated:
From the % Aggregation/Inhibition Chart supplied in the packaged tubes
Using the Plateletworks calculation wheel
Using the formula below

Baseline Platelet Count ‐ Agonist Platelet Count x 100 = % Aggregation

Baseline Platelet Count

Aspirin and Clopidogrel Response

Aspirin Response Analysis: Cartridge‐Based Platelet
Testing: Cartridge‐Based Platelet
Aggregation Aggregation

VerifyNow ﴾Accumetrics﴿ is a point‐of‐care platelet aggregometer that

uses single‐use test cartridges. Each cartridge contains fibrinogen‐coated
beads, platelet agonist, preservative, and buffer. The patient sample is
3.2% citrated whole blood, which is automatically dispensed from the
blood collection tube into the assay device by the instrument; no blood
handling is required by the user. The instrument controls assay
sequencing, establishes the assay temperature, controls the reagent
sample mixing for the required duration, determines the degree of
platelet function, displays the results and status information to the user,
and performs self‐diagnostics. The quality control ﴾QC﴿ system includes an
electronic QC, an assay device internal control, and two levels of external,
wet controls.
The VerifyNow Aspirin test is an optical method that utilizes arachidonic
Hover over the image to zoom in.
acid as the agonist to activate platelets. Agonist‐induced platelet
aggregation is enhanced by the use of fibrinogen‐coated beads and
measured as an increase in light transmission. The instrument
automatically calculates the results and expresses them as aspirin reaction
units. A cut‐off value is used to determine whether a patient has a sub‐
optimal response to aspirin therapy.

Aspirin and Clopidogrel Response

Clopidogrel Response Analysis: P2Y12 Analysis Testing: Cartridge‐Based Platelet
Aggregation

P2Y12 is an adenosine diphosphate ﴾ADP﴿ receptor located on platelet cell membranes. Clopidogrel functions by inhibiting this receptor. A P2Y12 assay
measures the inhibition of platelet function due to clopidogrel ingestion.
The VerifyNow™ P2Y12 assay ﴾Accumetrics﴿ is an another automated platelet aggregation test. It measures the level of platelet P2Y12 receptor inhibition.
The testing system employs single‐use cartridges so that the test can be performed at the point‐of‐care as well as in the laboratory. Like the company's
aspirin response test, it measures platelet‐induced aggregation as an increase in light transmittance. The required specimen is citrated whole blood. ADP is
used as the agonist in this test. The results are reported in P2Y12 Reaction Units ﴾PRU﴿.

Aspirin and Clopidogrel Response

Aspirin Response Analysis: Shear Flow Technology Testing: Other Aspirin Response
Tests

The PFA‐100 analyzer, produced by Siemens Healthcare Diagnostics, is used to evaluate platelet function. Platelet adhesion and aggregation are induced
at a high shear rate that simulates real platelet aggregation in vivo within smaller vessels.
An anticoagulated ﴾3.2% sodium citrate﴿ whole blood sample is added to one of two cartridges. Each cartridge contains a membrane coated with collagen
and an agonist; collagen/epinephrine ﴾CEPI﴿ is in one cartridge and collagen/ADP ﴾CADP﴿ is in the other. Platelets are activated and adhere to the
membrane and cause a thrombus to form within a tiny aperature in the middle of each membrane. When the membrane is occluded with aggregated
platelets, the timer stops. The time that it takes for complete occlusion to occur is called the closure time ﴾CT﴿ and is the measure of platelet activity. The CT
is recorded and compared to reference intervals. A prolonged CT with CEPI only indicates the presence of aspirin or aspirin‐like products such as non‐
steroidal anti‐inflammatory medications. CEPI‐induced CT should be prolonged when the patient is taking aspirin. If the result shows a lack of inhibition
with epinephrine and collagen, aspirin resistance may be indicated. If the CT is prolonged with both the CEPI and the CADP cartridges, it suggests platelet
dysfunction, and confirmatory testing would be needed.
Quality control for this instrument includes reagent QC utilized with normal patient samples each time an aggregation study is performed ﴾according to
good laboratory practice guidelines﴿. The manufacturer recommends analyzing QC with normal patient samples with each new shipment of reagents.

The PFA‐100 methodology animation is used with permission of the Platelet Research Laboratory and Siemens Healthcare Diagnostics.

View this course online to access video content.

Aspirin and Clopidogrel Response

Aspirin Response Analysis: Urinary 11‐ Testing: Other Aspirin Response
Dehydrothromboxane B2 for Aspirin Resistance Tests
View this course online to access video content.

Aspirin and Clopidogrel Response

Aspirin Response Analysis: Urinary 11‐ Testing: Other Aspirin Response
Dehydrothromboxane B2 for Aspirin Resistance Tests

There are alternative aspirin‐specific methodologies as well that test for resistance. For example, the 11‐dehydrothromboxane B2 ﴾11‐dTXB2﴿test. This
compound is a stable, inactive metabolite of thromboxane A2 and an in vivo indicator of platelet activity.
If 11‐dTXB2 is elevated in a urine sample from a patient receiving antiplatelet aspirin therapy, aspirin resistance may be indicated, since aspirin is intended
to inhibit thromboxane A2 production. The elevated level of 11‐dTXB2 indicates a continued hypercoagulable state and the patient may be at increased
risk for an ischemic cardiovascular event. If the test result is elevated, alternative anti‐thrombotic or antiplatelet therapies should be considered.
The AspirinWorks® test system is an enzyme‐linked immunosorbent assay ﴾ELISA﴿ for 11‐dTXB2. This test system will be discussed next.

Aspirin and Clopidogrel Response

Aspirin Response Analysis: AspirinWorks Analysis
Testing: Other Aspirin Response
Tests

AspirinWorks is an FDA‐approved kit that is used to determine aspirin effect. The test measures the thromboxane metabolite, 11‐dehydrothromboxane B2
﴾11‐dhTxB2﴿ in a urine sample.
Purified mouse monoclonal antibody and purified 11‐dhTxB2 conjugated to alkaline phosphatase compete for binding with the 11‐dhTxB2 present in the
urine sample. The intensity of color development following addition of chromogenic substrate is inversely proportional to the concentration of 11‐dhTxB2
in the urine sample. Results ﴾pg/mL﴿ are calculated against a reference curve. Final patient results are normalized by urine creatinine concentration, as
measured by a separate assay. A low level of 11‐dhTxB2 in the urine is associated with successful aspirin therapy ﴾aspirin is working to reduce
thromboxane production﴿. High levels of the biomarker may mean that the dosage of aspirin is not effective or a patient is aspirin‐resistant.

Aspirin and Clopidogrel Response

Ungraded Practice Question Testing: Other Aspirin Response
Tests

A PFA‐100 method system is used to evaluate platelet function in a patient who is on aspirin therapy. Which of the following collagen/epinephrine closure
time results would show a possible aspirin resistance in this patient ﴾Reference interval <164 s﴿?

Please select the single best answer

250 s

<164 s

Collagen/epinephrine closure time would not be used to evaluate aspirin resistance.

Aspirin and Clopidogrel Response

Ungraded Practice Question
Testing: Other Aspirin Response
Tests

A PFA‐100 method system is used to evaluate platelet function in a patient who is on aspirin therapy. Which of the following collagen/epinephrine closure
time results would show a possible aspirin resistance in this patient ﴾Reference interval <164 s﴿?

Please select the single best answer

250 s

<164 s

Collagen/epinephrine closure time would not be used to evaluate aspirin resistance.

Feedback

The correct answer is <164 s, if the reference interval is <164 s. Collagen/epinephrine closure time should be prolonged when the patient is taking
aspirin. If the CEPI cartridge result is not prolonged, aspirin resistance is possible.

Aspirin and Clopidogrel Response

Ungraded Practice Question Testing: Other Aspirin Response
 Aspirin and Clopidogrel Response
Ungraded Practice Question Testing: Other Aspirin Response
Tests

The elevation of which of the following substances in a urine sample may indicate a decreased response to aspirin therapy and an increased potential for
a future ischemic cardiovascular event in a high‐risk individual?

Please select the single best answer

Platelets

Cyclooxygenase‐1 enzyme ﴾COX‐1﴿

11‐dehydrothromboxane B2 ﴾11‐dTXB2﴿

Aspirin

Aspirin and Clopidogrel Response

Ungraded Practice Question Testing: Other Aspirin Response
Tests

The elevation of which of the following substances in a urine sample may indicate a decreased response to aspirin therapy and an increased potential for
a future ischemic cardiovascular event in a high‐risk individual?

Please select the single best answer

Platelets

Cyclooxygenase‐1 enzyme ﴾COX‐1﴿

11‐dehydrothromboxane B2 ﴾11‐dTXB2﴿

Aspirin

Feedback

The correct answer is 11‐dTXB2. 11‐dTXB2 is a stable, inactive metabolite of thromboxane A2 and an in vivo indicator of platelet activity. If 11‐dTXB2 is
elevated in a urine sample from a patient receiving antiplatelet aspirin therapy, aspirin resistance may be indicated. The elevated level of 11‐dTXB2
indicates a continued hypercoagulable state and the patient may be at increased risk for an ischemic cardiovascular event.

Aspirin and Clopidogrel Response

Additional Clopidogrel Response Tests Testing: Other Clopidogrel
Response Tests
In addition to the methodologies already discussed, there are other available options for testing Clopidogrel resistance, specifically. Two methods will be
outlined, including thromboelastography and CYP2C19 genotyping.

Aspirin and Clopidogrel Response

Clopidogrel Response Analysis: Thromboelastography Testing: Other Clopidogrel
Response Tests

Thromboelastography ﴾TEG﴿ is an assay that measures the visco‐elastic properties of whole blood clot formation under low shear stress. This testing is
normally used for surgical purposes to assess the quality of clot formation. However, a modified TEG method can be used to assess an individual's
response to antiplatelet therapy.
Agonists are added to a whole blood sample to begin the thrombotic process. The instrument has a pin attached to a tension wire which, when lowered
into a rotating cup, causes thrombi formation. The thrombi create a tension on the wire, which is converted into electrical signals that create graphical and
numerical outputs in a curve plot by the analyzer. When testing for clopidogrel response, the test is run with ADP‐, fibrin‐, and kaolin‐coated cups.
Evaluations of patient results are compared to the manufacturer's reagents for each system. Typically, a lack of inhibition of thrombus formation would
indicate resistance to clopidogrel. A cut‐off value that determines non‐responsiveness ﴾resistance﴿ to antiplatelet therapy must be established or verified
by the testing laboratory.
Thromboelastography devices such as the TEG® and RoTem® provide point‐of‐care monitoring of patient coagulopathic status. When clopidogrel
response is in question, TEG‐platelet mapping ﴾a modification of TEG﴿ can also be used to quantify the effect of this antiplatelet drug in patients.
numerical outputs in a curve plot by the analyzer. When testing for clopidogrel response, the test is run with ADP‐, fibrin‐, and kaolin‐coated cups.
Evaluations of patient results are compared to the manufacturer's reagents for each system. Typically, a lack of inhibition of thrombus formation would
indicate resistance to clopidogrel. A cut‐off value that determines non‐responsiveness ﴾resistance﴿ to antiplatelet therapy must be established or verified
by the testing laboratory.
Thromboelastography devices such as the TEG® and RoTem® provide point‐of‐care monitoring of patient coagulopathic status. When clopidogrel
response is in question, TEG‐platelet mapping ﴾a modification of TEG﴿ can also be used to quantify the effect of this antiplatelet drug in patients.

Aspirin and Clopidogrel Response

CYP2C19 Genotyping for Clopidogrel Testing: Other Clopidogrel
Response Tests

CYP2C19 is a drug‐metabolizing enzyme associated with clopidogrel metabolism. CYP2C19 is known to produce genetic variations/mutations, which can
affect the disposition of clopidogrel and therefore, the way people possessing the enzyme‐mutations respond to the drug. CYP2C19 can be evaluated
using either functional or genetic assays.
Certain common variants in the CYP2C19 gene are associated with decreased enzymatic activity and poor metabolism of clopidogrel, leading to a low
level of active drug and potentially ineffective treatment for those possessing the variant. If CYP2C19 genotyping identifies one of these genetic variations,
the physician may need to adjust the dosage of clopidogrel or prescribe an alternative therapy.
Although research has demonstrated that CYP2C19 variation contributes to a person's lack of responsiveness to clopidogrel, there is still a relatively small
demand for testing at this point. This may change as algorithms are updated and if research studies prove the value‐added benefit of these results.

Aspirin and Clopidogrel Response

Ungraded Practice Question Testing: Other Clopidogrel
Response Tests

Which platelet aggregometry methodology measures thrombi creation by tension on a wire which is measured in a curve plot by the analyzer?

Please select the single best answer

In vitro bleeding time assay

Optical Aggregometry

Thromboelastography

Urinary 11‐dehydro thromboxane B2 assay

Aspirin and Clopidogrel Response

Ungraded Practice Question Testing: Other Clopidogrel
Response Tests

Which platelet aggregometry methodology measures thrombi creation by tension on a wire which is measured in a curve plot by the analyzer?

Please select the single best answer

In vitro bleeding time assay

Optical Aggregometry

Thromboelastography

Urinary 11‐dehydro thromboxane B2 assay

Feedback

The correct answer is thromboelastography. This instrument has a pin attached to a tension wire, that when lowered into a rotating cup, causes thrombi
formation. The thrombi create a tension on the wire which is measured in a curve plot by the analyzer. When testing for clopidogrel response, the test is
run with ADP‐, fibrin‐, and kaolin‐coated cups.

Treatment for Antiplatelet

Treatment Alternatives When Aspirin or Clopidogrel Non‐ Medication Resistant Patients
responsiveness is Detected

Platelet function assays may be ordered to monitor therapy with antiplatelet medications. Patients whose test results indicate possible aspirin or
clopidogrel non‐responsiveness may need an adjustment to the dosage of the current medication or an alternative anticoagulant therapy. For example, if
 Treatment for Antiplatelet
Treatment Alternatives When Aspirin or Clopidogrel Non‐ Medication Resistant Patients
responsiveness is Detected

Platelet function assays may be ordered to monitor therapy with antiplatelet medications. Patients whose test results indicate possible aspirin or
clopidogrel non‐responsiveness may need an adjustment to the dosage of the current medication or an alternative anticoagulant therapy. For example, if
aspirin does not appear to be effective, the physician may prescribe clopidogrel instead. Patients who do not exhibit a satisfactory response to clopidogrel
therapy may be prescribed alternative antiplatelet medications such as prasugrel, ticagrelor, tirobifan or other P2Y12 receptor inhibitors.
Research studies and clinical trials continue to provide more clear‐cut recommendations regarding antiplatelet medication response testing, interpretation
of results, and appropriate actions based on the results. The clinical laboratory's role will most likely expand, if future studies conclude that testing for
individual response to these medications improves patient outcomes.

References
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