Platelet Function Tests

and Quality Management

연세의대 송재우
Platelet Adhesion and Activation

Rivera J et al. Haematologica 2009

Platelet Aggregation
Aggregometry

• Conventional methods: light transmission

– Gold standard
– Standardization of sample collection, processing and analysis
Aggregometry

• Conventional methods: light transmission

– No guideline of test interpretation or diagnostic criteria (ISTH SSC Platelet
Physiology Sub-committee guideline?)
Aggregometry

• Conventional methods: impedance

– Relatively little experience
– Hemoglobin?
– Maintenance?

Multiplate LTA

Test time 10 mins 1~ hrs

Sample volume 300 µl ~ 10 ml

Sample type whole blood PRP

Platelet Secretion

• Soluble activation markers (PPP)

– Platelet factor 4

– β-thromboglobulin

– Thromboxane B2 (urine)

– Serotonin uptake/release

– Soluble P-selectin

• Lumiaggregometer
– Bioluminescent determination ATP
POC Aggregometry: PFA
• Other detection methods of platelet aggregation?
• PFA
– Screening for VWD
– Measuring the effect of medications affecting platelet function (aspirin, DDAVP, Abciximab)
– Detection and diagnosis of acquired and congenital platelets dysfunctions

Fressinaud et al. Blood 1998

Mammen et al. Semin Thromb Hemost 1998

Clinical usefulness of LTA

• Bleeding disorders
• Thrombosis or antiplatelet therapy not
indicated for clinical purpose

• Any applications for research

External Quality Assurance (EQA)
Recent Initiatives from EQA Schemes

Recent Initiatives from Expert Groups

Expert Group Initiative Areas Covered by Initiative Main Findings

and References

CAP, RCPA EQA challenges Wet challenges using locally Confirmed that EQA for PFA-100 is feasible.
Hematology QAP for the PFA-100 collected blood with EQA provided Provided estimates of cross-laboratory
material and posttest analysis to precision and accuracy of testing for
assess precision and both normal and abnormal test results.
accuracy of PFA-100 testing
CLSI Approved Covers the pretest phase of patient Provides guidelines on platelet function
guideline for preparation, specimen collection, testing by diagnostic laboratories.
platelet and sample processing, Intended use is by clinicians, hospital
function testing establishment of reference intervals, and reference laboratorians, manufacturers,
results reporting, assay validation, and regulatory agencies. Guideline is
and troubleshooting, plus aspects of not intended to guide the performance of
quality control in relation to platelet global hemostasis tests, platelet counting,
function testing by aggregometry flow cytometry, point-of-care tests, test
(both IQC and EQA) interpretation, or therapy.
ISTH (LTA working International Evaluation of how platelet function Wide range of cross-laboratory test practice
group of the patterns-of- tests are performed in evidence. Data can be used to help
Platelet practice by diagnostic laboratories to gather drive improvements in testing (by
Physiology SSC) survey information on test practice, consensus and expert review).
including consensus of practice
ISTH (Platelet Publication of Platelet function testing for Provides guidelines on platelet function
Physiology SSC) guidelines. evaluating aspirin resistance; testing by diagnostic laboratories for
Additional assessment of platelet function specific circumstances.
guidelines under using the PFA-100; guidelines for
development. the performance of SEMINARS
LTA IN THROMBOSIS AND HEMOSTASIS/VOLUME 35, NUMBER 2 2009
General

• Validation of an instrument and competency of operators

minimum of 10 samples in duplicate and calculating the CV

• Linearity < dose response data of agonists

• New reagents: parallel and normal response check

• Each aggregation study: normal platelet control and chart

Pre-analytical variables

• Blood samples after a short rest period (median 7, 2–8)

• Refrain from smoking for at least 30 min (median 7, 5–8)
• Abstain from caffeine for at least 2 h (median 7, 3–8)
• Drug record (median 7, 7–9)
• Drugs reversibly inhibiting platelet (NSAIDs) stopped at least
3 days (median 8, 7–9)
• Drugs irreversibly inhibiting platelet (aspirin, thienopyridines)
stopped at least 10 days (median 8, 8–9)
• Uncertain
– fasting subjects (median 5, 3–8)
– any drug stopped (median 4, 1–8)
Specimen Collection

• Evacuated tube systems ≈ plastic syringes

– 19 – 21 gauge

– >19 gauge: traumatic and adverse hemostatic effect

– < 22 gauge: prolonged blood collection, hemolysis and platelet activation

– Syringe: needle removed → transfer (on the side wall)

• End-over-end inversion x 3 – 6

• 23 vs 21 gauge (children) needle with no significant differences

Anticoagulants

• Sodium Citrate
– Recommended anticoagulant (105 - 109 mmol/L, 3.2%)

– Effective calcium concentrations: ~40 μmol (109 mmol/L), <5 μmol (129 mmol/L)

– Sensitive to citrate content: ADP, epinephrine, collagen

• Anticoagulants to Avoid
– EDTA: chelating tenfold more calcium

– Heparin: thrombin inhibition, ADP response↑, response to epinephrine/collagen↓

– ACD: pH 6.5 PRP (no aggregate below a pH 6.4)

– PPACK: inhibitor of α-thrombin, effective calcium concentration↑

– Hirudin: GPIIb-IIIa (αIIbβ3) inhibitor study, MEA

Blood collection

• Minimal or no venostasis (median 8, 7–9)

• Needle <= 21 gauge (median 8, 7–9)
• Polypropylene or siliconized glass tubes (median 9, 8–9)
• Buffered anticoagulant (stable pH) (median 7, 3–8)
• 109 mM sodium citrate, buffered anticoagulant
(median 8, 5–9)
• 129 mM sodium citrate, buffered anticoagulant
(median 7, 4–9)
• The first 3–4 mL of blood discarded (median 8, 5–9)
• Under-filled tubes to be used to exclude severe platelet function disorders
(Glanzmann thrombasthenia, Bernard-Soulier syndrome) (median 7, 5–8)
Specimen Hematocrits

• Higher Hct: increased mean

threshold value for agonists
• Citrate adjustment
Specimen Transport

• Room temperature (20 - 25 °C)

– No exposure to cold (refrigeration, ice pack, winter) or heat (summer)

• Avoid pneumatic tube systems

• Upright tube position

• No vibration, shaking, agitation (hemolysis, platelet activation)

• Container of PRP
– To minimize pH elevation due to CO2 loss →

– Limited surface area-to-volume ratio, capped

Preparation of PRP and PPP

• Recommended centrifugation
– PRP: 170g x 15 minutes

– PPP: 1500 g x 15 minutes~ at RT

– Modification for special condition (large platelets, low platelet counts)

• Standardized platelet count of PRP: development of normal values

and interpretation guideline
– Recommended platelet count: 200 to 250 x 109/L

– Platelet count adjusted with autologous PPP

– platelet granule release during PPP preparation

– Physiologic buffer is an acceptable alternative

Preparation of PRP and PPP

• Room temperature for 15 min before centrifugation

(median 8, 6–8)
• Centrifuging blood samples at 200g x 10 min
(median 8, 5–9)
– Contamination of PRP
– Platelet reactivity
• Centrifuging at RT (median 8, 8–9)
• Centrifugation without brake (median 9, 8–9)
• PRP prepared by sedimentation for samples with very large
platelets (median 8, 5–9)
• PPP prepared by centrifuging at RT, 1500g for 15 min
(median 8, 8–9)
Interfering Substances

• Lipemia: effects on baseline turbidity

• Hemolysis
– Release of nucleotides → activation/desensitization (ADP) of platelets,
falsely depressed aggregation response

– braking of centrifuge, faulty pipetting

• Icteric specimen
– Depressed response by bilirubin > 2.4mg/dL
Assessment of PRP Quality

• Discard grossly hemolyzed samples (median 9, range 7–9)

• Lipemic sample included in the report (median 8, range 2–8)
• Check the platelet count of PRP (median 9, range 8–9)
• Possible inaccurate results with PRP platelet < 150 × 109/L (median 8,
5–9)
• PRP with low platelet counts to exclude severe platelet disorders
(Glanzmann thrombasthenia, Bernard-Soulier syndrome, VWD 2B,
pseudo-VWD) (median 8, 5–9)
• PRP platelet count should NOT be adjusted with autologous PPP
(median 8, 3–9)
Sample Storage

• Room temperature: more sensitive (ADP) than stored at 37°C

• Cold change
– Contraction, rounding, granular, microtubular loss, loss of plasma VWF
– Reversible within one hour
– Spontaneous aggregation with cold storage
• 37 °C
– Response loss
• Age of Sample
– Within 4 hours after platelet donation (change in pH)
– Initial (30 min) refractoriness (epinephrine) and subsequent gain of function
• PRP at RT and tested within 2 – 4 hours
• Significant response loss in 4 – 6 hours
Methodology

• Including a normal control (median 8, 7–9)

• PRP samples sit at RT for 15 min (median 7, 7–9)
• PRP used to set 0% light transmission (median 9, 8–9)
• Autologous PPP to set 100% light transmission (median 9, 8–9)
• Stirring at 1000 rpm (median 8, 8–9)

• Before adding an agonist baseline tracings > 1 min (median 8, 7–9)

• Consistent agonist volumes (< 10% of sample volume)
(median 8, 7–9)
• Minimum of 3 min after adding an agonist (median 8, 7–9)
• Minimum of 5 min if maximal aggregation not reached by 3 min with
control samples (median 8, 8–9)
• Minimum of 10 min if maximal aggregation not reached by 5 min with
control samples (median 8, 5–9)

• Tests completed within 4 h after blood sampling (median 8, 7–9)

Agonists

• ADP, epinephrine: primary and secondary wave

– primary wave: agonist – receptor

– Secondary wave: granular secretion

• Threshold titration
– Minimal concentration to induce the secondary wave

– Agonist concentrations to measure platelet secretion

• ADP (0.5 - 10 μmol/L, threshold 1 - 7.5 μmol/L)

– Shape change, (sharp deflection below the baseline)
Agonists

• Arachidonic acid (0.5 - 1.6 mmol/L.)

– U46619 (1 - 2 μmol/L): thromboxane derivative insensitive to aspirin

• Collagen (1 - 5 μg/mL,)
– Type I fibrillar collagen

– 1 - 2 μg/mL: low dose sensitive to aspirin

– 4 – 5 μg/mL: high dose insensitive to aspirin

– Platelet shape change. granule secretion independent of aggregation

• Epinephrine (0.5 - 10 μmol/L, threshold)

– No shape change

– No response in a subsets of healthy individuals

Agonists

• Ristocetin (RIPA)
– Mediates interaction between VWF and platelet (GPIbα)

– High-Dose RIPA (0.8 - 1.5 mg/mL)

– von Willebrand disease (Normalized by cryoprecipitate), Bernard-Soulier

syndrome (not normalized)

– Aspirin effect: aggregation-disaggregation (saw-tooth)

• Low-Dose RIPA (< 0.6 mg/mL)

– No normal response

– VWD type 2B or pseudo-VWD responsive

– VWD 2B normalized by cryoprecipitate, no change with pseudo-VWD

Other Agonists

• Thrombin
– α-thrombin: clot formation

– γ-thrombin (missing exosite I fibrinogen binding region)

– γ-thrombin: PAR1/4

– Thrombin receptor evaluation

• PAR1 (SFLLRN/TFLLRN)

• PAR4 (AYPGKF.
Agonists

• ADP: 2 μM (median 7, 4–9) → higher conc (median 8, 8–9)

• Epinephrine: 5 μM (median 8, 5–9) → higher conc (median 8, 6–9)
• Collagen: Low concentration of collagen (e.g. 2 μg/mL) (median 8, 5–9) → higher
conc (median 8, 6–9)
• PAR1-AP: 10 μM (median 7, 3–9) → higher conc (median 8, 5–9)
• U46619: 1 μM (median 7, 5–9) → higher conc (median 8, 4–9)
• Arachidonic acid: 1 mM (median 8, 6–9) → higher conc (median 7, 4–9)
• Ristocetin: 1.2 mg/mL (median 8, 7–9)
→ ristocetin 0.5–0.7 mg/mL if normal (median 8, 7–9)
→ ristocetin 2 mg/mL if no response (median 7, 3–9)
Result Analysis
Evaluation and reporting of results

• Evaluation of aggregation tracing

– Presence of shape change (median 8, 8–9)
– Length of the lag phase (median 8, 5–9)
– Slope of aggregation (median 7, 3–9)
– Maximal amplitude or % aggregation (median 9, 8–9)
– End amplitude or % aggregation (median 8, 5–9)
– Deaggregation (median 8, 8–9)
– Visual examination of the aggregation tracings (median 9, 8–9)
– Secondary wave induced by epinephrine (median 7, 5–9)

• Studies completed more than 4 h → comment (median 8, 3–9)

• Reference intervals and test performance validation with each lot
of reagents (median 8, 7–9)
Whole Blood Impedance Aggregometry

• Anticoagulation: trisodium citrate (105 - 109 mmol/L, 3.2%),

hirudin (25 μg/mL, MEA)

• Transport and storage

– RT

– Pneumatic system not recommended

– Test within 3 hours

• Dilution
– Platelet count 100 - 1000 x 109/L: diluted (x 1/2) with saline

– < 100 x 109/L tested undiluted

– ADP: Needs platelet count > 225 x 109/L

Agonists

• Measured in ohms/AUC (MEA)

• ADP (5 - 20 μmol/L): delay for 20 - 30 mins following
venipuncture
• Arachidonic acid (0.5 - 1.0 mmol/L): aspirin effect (aggregation↓,
ATP secretion↓ with low concentrations (0.5 mmol/L)
• Collagen (1 - 5 μg/mL)
– Lag phase of up to 1 minute
– Aspirin effect at 1 - 2 μg/mL, normal response at 5 μg/mL
• Ristocetin (RIPA)
– High-Dose RIPA (1.0 mg/mL)
– Low-Dose RIPA (0.25 mg/mL)
• TRAP-6(32 μmol/L): MEA analysis
– aggregation insensitive to aspirin or clopidogrel
Methods

• Test whether or not the impedance probe is capable of detection

a change in electrical resistance by inserting the probe in a
cuvette with 1mL of prewarmed physiological saline and a stir bar

• Set the baseline at 0 ohms of aggregation and then set the 20-
ohm gain at 50% of the graph.

• When the baseline returns to 0 ohms, add 200 uL of water to the

sample- immediate drop of the impedence line from the baseline
(approximately 20%)

• If the impedence aggregation tracing jumps erratically during the

test- may be a problem with either a damaged or dirty probe,
follow the probe cleaning procedure.
Result Analysis
Flow and High Shear Devices

• Anticoagulant
– Sodium Citrate Anticoagulant 105 - 109 mmol/L (3.2%) or 129 mmol/L (3.8%)
– 3.8% greater sensitivity (at one hour) to aspirin effect

• Specimen Hematocrits
• Hct < 20% : no closure
• Hct > 50%: possible erratic measurements
Flow and High Shear Device

• Electronic mechanical check up (per shift)

• Parallel tests with blood drawn from healthy donor
– New batch
– significant change to the equipment
– Mean value of the duplicate within the reference interval
EQA: PFA-100/PFA-200

• Tubes containing either

– no additive
– GPIIb/IIIa inhibitor
– Citrate anticoagulated blood from healthy donor transferred
to EQA tubes
– PFA-100/PFA-200 tests

• Royal College of Pathologists of Australasia (RCPA) Hematology

Quality Assurance Program (QAP)
• U.S.-based College of American Pathologists (CAP)
EQA: PFA-100/PFA-200

Pavaloro E, Semin Thromb Hemost 2014

 PFA-100
( 2015 CAP PF1-A)
EQA to IQA: PFA-100/PFA-200
References
• Favaloro EJ and Bonar, External Quality Assessment/Proficiency Testing and
Internal Quality Control for the PFA-100 and PFA-200: An Update. Semin
Thromb Hemost. 2014;40:239.
• Cattaneo M, Cerlett C, Harrison P, Hayward C, Kenny D, Nugent D,
Recommendations for the standardization of light transmission aggregometry:
a consensus of the working party from the platelet physiology subcommittee
of SSC/ISTH. J Thromb Haemost. 2013;11:1183.
• Cattaneo M, Hayward C, Moffat K, Pugliano M, Liu Y, Michelson A, J Thromb
Haemost. 2009;7:1029.
• CLSI H58A, Platelet Function Testing by Aggregometry; Approved Guideline.
• Favaloro EJ. Internal Quality Control and External Quality Assurance of Platelet
Function Tests. Semin Thromb Hemost. 2009;35:139.