Platelet Aggregation Testing Online Training

Welcome to the Platelet Aggregation* Online Training course. The introduction of platelet aggregation testing on the Sysmex CS Systems provides your laboratory with: Increased automation during the testing process that frees technologists to perform other tasks   Increased standardization that provides your laboratory with consistency and quality in patient care   Delivery of excellent care to patients with demands for high-quality and accurate results along with standardization of quality of care * Product availability may vary from country to country and is subject to varying regulatory requirements. Please contact your local representative for availability. Sysmex is a registered trademark of Sysmex Corporation. All other trademarks and brands are the property of their respective owners.   Upon successful completion of this course, you will be able to: Describe the principle used in platelet aggregation testing List the clinical disease states that warrant the need for platelet aggregation testing Describe the sample processing steps of platelet aggregation testing Identify the various reagents (agonists) used by the Sysmex CS series analyzers Discuss the importance of the sample collection and preparation steps Congratulations. You have completed the Platelet Aggregation Testing Online Training course. Listed below are the key points that have been presented. Take time to review the material before you proceed to the final quiz. Describe the principle used in platelet aggregation testing The core principle behind the platelet aggregation test is based upon light transmission aggregometry which has been in use for more than 50 years and is considered the gold standard of testing. Light transmission aggregometry is based upon the principle that light transmission of a sample increases as platelets aggregate. Platelet Poor Plasma (PPP) is used as the 100% reference value Platelet Rich Plasma (PRP) is used as the 0% reference value prior to the addition of agonists The ability of various agonists to induce platelet activation is measured over time as the sample is continuously mixed at 37°C.  The amount and rate of the increase in light transmission are directly proportional to the reactivity of the platelets to the agonists. These changes in light transmission are recorded as a reaction curve plotted against time. Maximum aggregation rate (%) =  (initial light absorbance of PRP - minimum light absorbance of PRP)  x 100             (initial light absorbance of PRP - light absorbance of PPP) List the clinical disease states that warrant the need for platelet aggregation testing Clinical Need for Platelet Aggregation Testing includes: Deficiency of platelet aggregation/Bleeding disorders Glanzmann thrombasthenia Von Willebrand disease Bernard Soulier Syndrome Storage pool disease, etc.   Antiplatelet drug therapy Aspirin - Binding to cyclooxygenase (COX-1) ADP receptor inhibition(e.g. Clopidogrel, Ticlopidine etc.) - In ADP major receptor, there is P2Y1, P2Y12 Phosphodiesterase (PDE) inhibition drug (e.g. Cilostazol)   Potential risk of a diagnosis of thrombosis disease Describe the platelet aggregation testing workflow Key steps of the platelet aggregation workflow include: Sample collection and preparation Reagent preparation Sample processing Results review Discuss the importance of the sample collection and preparation steps Key criteria during sample collection includes: Patients should fast prior to blood collection to avoid lipemic samples Blood collection should be as smooth as possible to avoid contamination with tissue factor (discard any hemolytic samples) Minimize use of tourniquet Preferred needle size is 21 gauge Use only plastic or siliconized sample tubes with a citrate anticoagulant Mix tube gently after collection Key steps during sample preparation include: Centrifuge blood sample - centrifuge samples at room temperature for 10 minutes at 200 xg. No brake should be used. Separate PRP - be careful not to mix PRP with blood cells.  Collect sample up to 0.5 - 1.0 cm from blood cell layer. Centrifuge remaining blood - centrifuge at room temperature for 15 minutes at 1500 xg.  Use brake. Separate PPP - collect sample 0.5cm from blood cell layer. Measure PRP platelets - confirm platelet count is between 150,000 to 480,000 Store samples (PRP and PPP) at room temperature until used. Complete testing within 4 hours of sample collection. For more information about the sample collection please refer to ISTH and CLSI guidelines for platelet aggregation testing Platelet Rich Plasma (PRP) Contains platelets in suspension Light is unable to pass through Serves as 0% Transmission Reference value Addition of agonists induce platelet aggregation Suspension clears over time while sample is mixed at 37°C Changes in light transmission are recorded as reaction curve against time Platelet Poor Plasma (PPP) Contains few to no platelets in suspension Light passes through sample uninterrupted Used as 100% Transmission Reference value Identify the various reagents (agonists) used by the Sysmex CS series analyzers Reagents (agonists) include: ADP Collagen Epinephrine Arachidonic Acid Ristocetin Agonist types include: Weak - induce platelet aggregation without inducing secretion of platelet granules Strong - directly induce platelet aggregation, Thromboxane synthesis, and platelet granule secretion Purpose of the addition of agonists: As PRP is mixed with the various agonists, the platelets begin to aggregate Light transmission of the PRP changes Change in transmission is detected and measured at 660nm Maximal aggregation (%) is calculated by the maximal amount of change in the light transmission caused by the platelet aggregation which is induced by the agonist Describe the sample processing steps of platelet aggregation testing Key steps of sample processing on the Sysmex CS series analyzers: Prepare and set reagent Set the SB Cuvettes using the SB Set Tool Prepare and set PPP and PRP Order platelet aggregation assays Start measurement Results Review The automated results produce clear aggregation histograms (test reaction graphics) for interpretation* by clinicians so that they can provide an accurate diagnosis and ensure the highest quality of care for patients. *Refer to your Laboratory's Standard Operating Procedures for more specific information on the interpretation of results. Sample Collection and Preparation Reagent Preparation Sample Processing Results Review Key steps of sample processing on the Sysmex CS series analyzers: Prepare and set reagent Set the SB Cuvette using the SB Set tool Prepare and set PPP and PRP Order platelet aggregation assays Start measurement Sample Processing Sample Processing Instructions:Flash File:/content/generator/Course_90016670/SIM_PAGG_SampleProcessing_900x500/SIM_PAGG_SampleProcessing_900x500.swfHTML5 File:/content/generator/Course_90016670/SIM_PAGG_SampleProcessing_900x500/index.htmlPDF File: Reagents (agonists) include: ADP Collagen Epinephrine Arachidonic Acid Ristocetin Agonist Types include: Weak - induce platelet aggregation without inducing secretion of platelet granules Strong - directly induce platelet aggregation, Thromboxane synthesis, and platelet granule secretion   Reagent Preparation Reagent Preparation Slide NumberText BlocksCalloutsAudio ScriptImage File1Purpose of the addition of agonists: As PRP is mixed with the various agonists, the platelets begin to aggregate Light transmission of the PRP changes Change in transmission is detected and measured at 660nm Maximal aggregation (%) is calculated by the maximal amount of change in the light transmission caused by the platelet aggregation which is induced by the agonist Note:  Sysmex CS Series Analyzer Application sheets list detailed information for each assay. As we mentioned earlier, when agonists are mixed with a platelet rich plasma sample, the platelets begin to aggregate. As the platelets aggregate, the percentage of light transmission in the platelet rich sample begins to increase and this change is detected and measured at 660nm. The maximal aggregation percentage is then calculated for each sample. 2ADP Reagent Preparation: Allow reagent to equilibrate at room temperature Reconstitute with 0.625mL of distilled water to prepare a stock solution with a concentration of 160µmol/L Mix thoroughly and allow to sit for 30 minutes to obtain a homogeneous solution stock solution Dilute 1:10 with 0.9% NaCl in a 4mL sample cup to prepare a working solution with a concentration of 16µmol/L A final dilution of 1:8 in the reaction cuvette leads to a final concentration of 2µmol/L. Use ADP working solution within a day of preparation Storage and Shelf Life of Stock Solution: 18 to 25°C - Stable 24 hours 2 to 8°C - Stable 28 days ≤ - 20°C - Stable 2 months Onboard stability: 10 hoursNote: Refer to the instrument specific ADP Application guide and IFU for more specific reagent information.Once the ADP reagent has reached room temperature, reconstitute the reagent with 0.625 milliliters of distilled water. Mix thoroughly and allow the suspension to sit at room temperature for 30 minutes to achieve a homogenous stock solution. Next, dilute the reconstituted reagent 1:10 with 0.9% saline and dispense into a sample cup. It is important to note that the diluted reagents must be used immediately for testing3Collagen Reagent Preparation: Allow reagent to equilibrate at room temperature Reconstitute with 0.625mL of distilled water to prepare a stock solution with a concentration of 800µg/mL Mix thoroughly and allow to sit for 30 minutes to obtain a homogeneous stock solution Dilute 1:50 with Collagen diluent in a 4mL sample cup to prepare a working solution with a concentration of 16µg/mL A final dilution of 1:8 in the reaction cuvette leads to a final concentration of 2µg/mL Use Collagen working solution within a day of preparation Storage and Shelf Life of Stock Solution: 18 to 25°C - Stable 24 hours 2 to 8°C - Stable 28 days Do not freeze collagen solution Onboard stability: 10 hours  Note: Refer to the instrument specific Collagen Application guide and IFU for more specific reagent information.Collagen reconstitution follows a similar process as ADP; however, the homogenous suspension is diluted to a ratio of 1:50 with a collagen diluent in a 4mL sample cup for testing. It is important to note that unlike the other agonists, the collagen reagent may not be stored at frozen temperatures.4Ristocetin Reagent Preparation: Allow reagent to equilibrate at room temperature Reconstitute with 0.625mL of distilled water to prepare a stock solution with a concentration of 12mg/mL Mix thoroughly and allow to sit for 30 minutes to obtain a homogeneous stock solution Dilute 1:1.25 with 0.9% NaCl in a 4mL sample cup to prepare a working solution of 9.6mg/mL A final dilution of 1:8 in the reaction cuvette leads to a final concentration of 1.2mg/mL Use Ristocetin working solution within a day of preparation Storage and Shelf Life of Stock Solution: 18 to 25°C - Stable 8 hours 2 to 8°C - Stable 7 days ≤ -20°C - Stable 2 months Onboard stability: 10 hours  Note: Refer to the instrument specific Ristocetin Application guide and IFU for more specific reagent information.Once the Ristocetin reagent is reconstituted and has reached a homogenous suspension, it is diluted ratio 1:1.25 with 0.9% saline in a 4 mL sample cup for further testing.5Arachidonic acid Reagent Preparation: Allow reagent to equilibrate at room temperature Reconstitute with 0.625mL of distilled water to prepare a stock solution with a concentration of 12mmol/L Mix thoroughly and allow to sit for 30 minutes to obtain a homogeneous stock solution Dilute 1:1.5 with 0.9% NaCl in a 4mL sample cup to prepare a working solution with a concentration of 8mmol/L A final dilution of 1:8 in the reaction cuvette leads to a final concentration of 1mmol/L Use Arachidonic Acid working solution within a day of preparation Storage and Shelf Life of Stock Solution: 18 to 25°C - Stable 24 hours 2 to 8°C - Stable 7 days ≤ - 20°C - Stable 2 months Onboard stability: 10 hours  Note: Refer to the instrument specific Arachidonic acid Application guide and IFU for more specific reagent information.Arachidonic acid is reconstituted in a similar fashion to the other reagents and the homogenous suspension is then diluted ratio 1:1.5 with 0.9% saline in a 4 mL sample cup for further testing. 6Epinephrine Reagent Preparation: Allow reagent to equilibrate at room temperature Reconstitute with 0.625mL of distilled water to prepare a stock solution with a concentration of 800µmol/L Mix thoroughly and allow to sit for 30 minutes to obtain a homogeneous stock solution Dilute 1:20 with 0.9% NaCl in a 4mL sample cup to prepare a working solution with a concentration of 40µmol/L A final dilution of 1:8 in the reaction cuvette leads to a final concentration of 5µmol/L Use Epinephrine working solution within a day of preparation Storage and Shelf Life of Stock Solution: 18 to 25°C - Stable 24 hours 2 to 8°C - Stable 7 days ≤-20° - Stable 2 months Onboard stability: 10 hoursNote: Refer to the instrument specific Epinephrine Application guide and IFU for more specific reagent information.The reconstituted and homogenous Epinephrine suspension is diluted ratio 1:20 with 0.9% saline in a 4 mL sample cup for testing.7Summary of reconstitution, dilution, and stability of each agonist used in the Platelet Aggregation test.   ADP Collagen Epinephrine Arachidonic Acid Ristocetin Component 0.625* mL x3 0.625* mL x3 0.625* mL x3 0.625* mL x3 0.625* mL x3 Concentration 160 µmol/L 800µg/mL 800µmol/L 12 mmol/L 12 mg/mL Diluent 0.9% NaCl Collagen diluent 0.9% NaCl 0.9% NaCl 0.9% NaCl Dilution Ratio 1:10 1:50 1:20 1:1.5 1:1.25 Working Concentration 16 µmol/L 16 µg/mL 40 µmol/L 8.0 mmol/L 9.6 mg/mL Onboard Stability 10 hours 10 hours 10 hours 10 hours 10 hours Stock Solution Stability   2 - 8 ° 28 days 28 days 7 days 7 days 7 days 18 - 25° 24 hrs. 24 hrs. 24 hrs. 24 hrs. 8 hrs. -20° 2 months NA 2 months 2 months 2 months *Must reconstitute with 0.625mL of purified water when running Platelet Aggregation Test on Sysmex analyzers.  The table displayed here summarizes the dilution, concentration, and storage requirements for each of the five agonists we have discussed. The stability of each reagent onboard the system is ten hours. Key specimen criteria during sample collection Patients should fast prior to blood collection to avoid lipemic samples Blood collection should be as smooth as possible to avoid contamination with tissue factor (discard any hemolytic samples) Minimize use of tourniquet Preferred needle size is 21 gauge Use only plastic or siliconized sample tubes with a citrate anticoagulant Mix tube gently after collection   Key steps during sample preparation Centrifuge blood sample Separate PRP Centrifuge remaining blood Separate PPP Measure PRP sample to ensure adequate platelet count For more information about the sample collection please refer to ISTH and CLSI guidelines for platelet aggregation testing.   PRP versus PPP PRP versus PPP Tab TitleTextPlatelet Rich Plasma (PRP) Platelet Rich Plasma (PRP) - plasma containing platelets that is prepared by low centrifugal force Contains platelets in suspension Light is unable to pass through Serves as 0% Transmission Reference value prior to the addition of agonists Addition of agonists induce platelet aggregation Suspension clears over time while sample is mixed at 37°C Changes in light transmission are recorded as reaction curve against time Platelet Poor Plasma (PPP) Platelet Poor Plasma (PPP) - plasma that has been depleted of platelets through high centrifugal force Contains few to no platelets in suspension Light passes through sample uninterrupted Used as 100% Transmission Reference value Sample Preparation and Storage Sample Preparation and Storage Checklist TitleChecklist TypeChecklist ContentCentrifuge sampleHTML Centrifuge samples at 200 xg for 10 minutes at room temperature. Do NOT use a brake. Carefully remove sample from centrifuge when complete. Separate PRPHTML Confirm separation of PRP and collect the supernatant by pipette.  Seal the PRP tube and let stand at room temperature for 30 minutes. Note: Be careful not to mix PRP with blood cells.  Collect sample up to 0.5 - 1.0 cm from blood cell layer. Centrifuge remaining bloodHTML Centrifuge the remaining blood at 1500xg for 15 minutes at room temperature. Brake should be used. Separate PPP and Measure PRPHTML Confirm the separation of PPP and collect the supernatant using a plastic pipette. Collect sample 0.5cm from blood cell layer being careful not to disturb the red blood cell layer. Dispense required volume of PRP sample to measure platelet count. Confirm platelet count is between 150,000 to 480,000 (sufficient platelets must be present in order to produce a functional response that falls within the threshold limitations of the test) Store samplesHTML Store samples (PRP and PPP) at room temperature until testing. Complete testing within 4 hours of sample collection. Interfering Substances Interfering Substances Drugs that have the potential to interfere with platelet function include: Aspirin Anti-inflammatory drugs Specific anti-platelet drugs including Clopidogrel and Imidazole Antibiotics Anti-depressants Beta-blockers   Clinical Need for Platelet Aggregation Testing includes: Deficiency of platelet aggregation/Bleeding disorders   Glanzmann thrombasthenia Von Willebrand disease Bernard Soulier Syndrome Storage pool disease, etc.   Antiplatelet drug therapy   Aspirin - Binding to cyclooxygenase (COX-1) ADP receptor inhibition(e.g. Clopidogrel, Ticlopidine etc.) - In ADP major receptor, there is P2Y1, P2Y12 Phosphodiesterase (PDE) inhibition drug (e.g. Cilostazol)   Potential risk of a diagnosis of thrombosis disease   PPP PRP 100% Transmission 0% Transmission agonist stir magnet Time ⇒ % Transmission ⇒ Maximum aggregation rate (%) = (initial light absorbance of PRP - minimum light absorbance of PRP)  x 100          (initial light absorbance of PRP - light absorbance of PPP) PPP PRP The automated results produce clear aggregation histograms (test reaction graphics) for interpretation* of the results by clinicians so that they can provide an accurate diagnosis and ensure the highest quality of care for patients. *Refer to your Laboratory's Standard Operating Procedures for more specific information on the interpretation of results. Check Measurement Results Check Measurement Results Instructions:Flash File:/content/generator/Course_90016670/SIM_PAGG_CheckMeasurements_900x500_Rev/SIM_PAGG_CheckMeasurements_900x500_Rev.swfHTML5 File:/content/generator/Course_90016670/SIM_PAGG_CheckMeasurements_900x500_Rev/index.htmlPDF File: PPP High Error Troubleshooting: PPP High Error Element HTMLPPP High Error If PPP Absorbance result is high, PRP could have been set instead of PPP.   Sound File Audio ScriptAs we have already discussed, there are key steps during sample collection and processing, as well as, reagent preparation that can impact test results.  We also stressed the importance of the PPP and PRP samples for each patient.  If you encounter a higher than expected result for the PPP sample, it is possible that the PRP rather than PPP sample was set on the system by mistake.  Shown here is the error message that would display if the PPP sample yields a higher than expected result.