PFA-100® Analyzer Sample Processing

Welcome to the PFA-100® Analyzer Sample Processing online training course. Sample processing is where all the action takes place on the PFA-100® instrument from collecting samples, loading test cartridges, and evaluating closure time test results that measure platelet function. In this course, you’ll learn how to properly collect PFA-100® samples for testing. You’ll also learn about PFA-100® printing options that allow you to view stored data. In addition, you’ll learn how to evaluate and interpret test analysis patterns as well as identify and describe how certain medications and platelet disorders affect closure time results. NOTE: There is no audio in this course. Upon successful completion of this course, you will be able to: List PFA-100® sample collection requirements Demonstrate how to run patient samples on the PFA-100® analyzer Describer how to print PFA-100® stored data Evaluate and interpret PFA-100® test analysis patterns Identify and describe how certain medications and platelet disorders affect PFA-100® closure time results Congratulations. You have completed the PFA-100® Analyzer Sample Processing online training course. Listed below are the key learning objectives presented. List PFA-100® sample collection requirements. To obtain accurate closure time (CT) results, collect blood samples without causing any trauma to red blood cells and platelets that could accidentally initiate the clotting process. Use a 21-gauge or larger needle to draw blood directly into an evacuated plastic or siliconized glass tube or syringe containing 3.2% (0.105M) or 3.8% (0.129M) buffered sodium citrate (1-part anticoagulant to 9-parts blood). Discard the sample if hemolyzed or if venous collapse or stoppage of blood flow occurs during collection. Also, note the following: Store samples undisturbed at room temperature for up to 4 hours. Gently invert the tube by hand 3 to 4 times to ensure proper mixing with anticoagulant. DO NOT place tubes on a rocker or rotator while waiting to be tested. DO NOT refrigerate or centrifuge samples. Demonstrate how to run samples on the PFA-100® instrument. Sample processing options include: Running a single test on a patient sample by loading a Collagen/Epinephrine (COL/EPI) or Collagen/ADP (COL/ADP) test cartridge in cassette position A or B. Running a test in duplicate on a patient sample by loading the same type of test cartridge in cassette positions A and B. Running two different tests on a patient sample by loading one type of test cartridge in cassette position A and another type of test cartridge in cassette position B. Running a single test on two different patient samples by loading test cartridges in cassette positions A and B. Describe how to print PFA-100® stored data. The PFA-100® system is set to automatically print CT results after testing is complete. From the System Ready display, you can print the results of the last run, or from the Menus display, you can select options to print stored results or usage history. Evaluate and interpret PFA-100® test analysis patterns. Analysis patterns for CT results using COL/EPI and COL/ADP cartridges allow you to differentiate between platelet dysfunction due to a platelet inhibiting factor (such as Aspirin® or other aspirin-containing medication) versus platelet dysfunction due to a platelet disorder. For example, COL/EPI results are usually abnormal and COL/ADP results are normal for patients taking Aspirin® or other aspirin-containing medication. Both test cartridges yield abnormal results for patients with von Willebrand disease or Glanzmann thrombasthenia. Identify and describe how certain medications and platelet disorders affect PFA-100® closure time results. Several factors can affect PFA-100® closure time results: Aspirin® or other aspirin-containing medication can yield abnormal CT results using COL/EPI cartridges, but normal CT results using COL/ADP cartridges. Microthrombi can affect the PFA-100® test results and/or cause a cancellation of the test by the instrument due to the detection of a flow obstruction. Blood samples with high sedimentation rates may experience settling in position B while waiting to be tested in sequence with position A, which could potentially affect CT results. Patients with low hematocrit levels <35% can yield prolonged CT results due the inability of red blood cells to push platelets toward the periphery of a vessel to form an effective platelet plug, which is part of the mechanism measured by the PFA-100® system. Patients with low platelet counts <150,000/mL can yield prolonged CT results because in vivo platelet plug formation requires normal functioning platelets as well as adequate numbers of platelets. Certain fatty acids and lipids found in various human diets are known to inhibit platelet function. Hemolyzed blood can interfere with PFA-100® test results by decreasing the hematocrit and by releasing ADP, which can activate platelets. If the instrument is configured to query for patient ID, you can enter unique patient IDs for each sample. If not configured, the instrument automatically assigns an accession number. Sample processing options include: Running a single test by loading a test cartridge in cassette position A or B Running a test in duplicate by loading the same type of test cartridge in cassette positions A and B Running two different tests by loading one type test cartridge in position A and another type test cartridge in position B Running a single test on two different patient samples by loading test cartridges in positions A and B Patient samples must be stored at room temperature and are stable for up to 4 hours prior to testing. Make sure test cartridges have reached room temperature before using (approximately 15 minutes). To minimize the risk of trapping an air bubble in the test cartridge, place the tip of the pipette against an inside corner of the opening while you slowly pipette sample into the reservoir. Pneumatic tube transport of samples for platelet function tests may adversely affect results. Hand delivery is recommended to ensure reliable platelet function test results.  Single Test Learn how to run a single test. Checklist TitleChecklist TypeChecklist ContentLoad CassetteHTML For this test, let’s use a Collagen/Epinephrine (COL/EPI) test cartridge. You can load a test cartridge into cassette positions A or B. Push the COL/EPI cartridge into the cassette until it snaps securely in place. The PFA-100® automatically determines the test cartridge type before testing the sample. Invert TubeHTML Gently invert the collection tube 3-4 times by hand and then pipette 800 µL of sample into the sample reservoir opening of the test cartridge. Be sure to place the pipette tip along the wall of the sample reservoir to avoid pipetting bubbles. Load Cartridge in Incubation WellHTML Place the cassette with the test cartridge into the incubation well of the instrument so that the cassette is flush to the carousel surface. Be sure that you do not apply pressure to the sample reservoir opening when inserting the cassette. Start Sample ProcessingHTML To start sample processing, from the System Ready display, press the softkey located next to Run. The carousel will rotate to the instrument’s interior. The sample starts warming in the carousel’s incubation wells. Enter Patient IDHTML Because the PFA-100® is configured to query for patient ID, the system prompts you to enter the patient ID. Use the keypad to enter the patient ID. Patient IDs can have a maximum of 12 characters. If you make an error, press the < symbol to back up one space. You can also use the optional bar code reader to enter the patient ID. Start AnalysisHTML To start the analysis, press the softkey next to Run. During test, the LCD shows the processing stages (incubating, testing sample A, testing sample B) as applicable.  Print ResultsHTML When testing is complete, the System Ready display reappears and the instrument prints out the COL/EPI closure time result. The printout will also display the test cartridge type, patient ID, and status messages, if the system detected any events or conditions during the analysis. Remove CassetteHTML After testing, remove the cassette carefully from the carousel. Holding the cassette in one hand, remove the test cartridge by gently pulling the bottom of the cartridge toward you until it unsnaps. Be sure to dispose of the test cartridge in a suitable biohazard waste container. Duplicate Tests Learn how to run duplicate tests. Checklist TitleChecklist TypeChecklist ContentLoad CassetteHTML Place two of the same type of test cartridges in cassette positions A and B. For this test, let’s use COL/EPI test cartridges. Push the COL/EPI cartridges into the cassette until they snap securely in place.  Invert TubeHTML Gently invert the collection tube 3-4 times by hand and then pipette 800 µL of sample into the sample reservoir opening of each test cartridge.  Be sure to place the pipette tip along the wall of the sample reservoir to avoid pipetting bubbles. Load Cartridge in Incubation WellHTML Place the cassette with the test cartridge(s) into the incubation well(s) of the instrument so that the cassette is flush to the carousel surface. Remember not to apply pressure to the sample reservoir opening.  Start Sample ProcessingHTML To start sample processing, from the System Ready display, press the softkey located next to Run. The carousel will rotate to the instrument’s interior.  Enter Patient IDHTML Because the PFA-100® is configured to query for patient ID, the system prompts you to enter the patient ID. If two test cartridges are loaded in positions A and B, the PFA-100® first prompts you for the ID of the sample loaded in position A.  Start AnalysisHTML To start the analysis, press the softkey located next to Run Duplicate. During testing, the LCD shows the test processing stage ¾ incubating, testing sample A, testing sample B, as applicable. As long as you select Run Duplicate, the only difference between running a sample in duplicate and running two different tests on the same sample is to load either the same type of test cartridge or different types of test cartridges in cassette positions A and B.  Print ResultsHTML When testing is complete, the System Ready display reappears and the instrument prints out the COL/EPI closure time results. Note the CT results, CV%, and mean value. The printout will also display the test cartridge type, patient ID, and status messages, if the system detected any events or conditions during the analysis. Remove CassetteHTML After testing, remove the cassette carefully from the carousel. Holding the cassette in one hand, remove each test cartridge by gently pulling the bottom of the cartridge toward you until it unsnaps. Be sure to dispose of the test cartridge in a suitable biohazard waste container. Tests for Different Patients Learn how to run different tests. Checklist TitleChecklist TypeChecklist ContentLoad CassetteHTML Push in the COL/EPI cartridges in cassette positions A and B until they snap securely in place.   Invert TubeHTML Gently invert the collection tube of the first patient sample 3-4 times by hand and then pipette 800 µL of sample into the sample reservoir opening of the test cartridge in cassette position A. Repeat this process using the second patient sample to load the sample reservoir for cassette position B. Place the pipette tip along the wall of the sample reservoir to avoid pipetting bubbles. Load Cartridge in Incubation WellHTML Place the cassette with the test cartridges into the incubation wells of the instrument so that the cassette is flush to the carousel surface. Remember not to apply pressure to the sample reservoir opening.  Start Sample ProcessingHTML To start sample processing, from the System Ready display, press the softkey located next to Run. The carousel will rotate to the instrument’s interior.  Enter Patient ID Position AHTML Because the PFA-100® is configured to query for patient ID, the system prompts you to enter the patient ID. Using the keypad, enter the first patient sample ID in position A. Remember that by default, the system first prompts you for the sample ID for position A.  Enter Patient ID Position BHTML To enter a new sample ID for position B, press Run to “tell” the instrument that the test cartridge in position B will have a different sample ID than the sample ID entered for position A. The system will now prompt you to enter the patient ID for the sample in position B. Press the softkey located next to Run to start the analysis for both samples. Start AnalysisHTML  During testing, the LCD shows the test processing stage - incubating, testing sample A, testing sample B, as applicable. Print ResultsHTML The printout displays the CT results, test cartridge types, patient IDs, and status messages, if the system detected any events or conditions during the analysis. Remove CartridgeHTML After testing, remove the cassette carefully from the carousel. Holding the cassette in one hand, remove each test cartridge by gently pulling the bottom of the cartridge toward you until it unsnaps. Be sure to dispose of the test cartridge in a suitable biohazard waste container. The PFA-100® system is set to automatically print closure time (CT) results after testing is complete. You can also print the results of the last run, print stored results, and print usage history.  Print Results Learn how to print results. Tab TitleTextPrint Last Run1. From the System Ready display, press the softkey located next to Print Last Run.Print Stored Results From the System Ready display, press the softkey located next to Menus On the Menus display, select Print Log, number 4 on the keypad Select number 1 on the keypad to select Patient Results Use the keypad to enter the number of patient results to be printed (maximum 20) Press the softkey located next to Enter The system will begin printing the results of previous tests starting with the most recent one. Events or conditions (Status Messages) detected during the test run will be displayed below the printed test result Press the softkey located next to Previous Screen twice to return to the System Ready display Print Usage History From the System Ready display, press the softkey located next to Menus On the Menus display, select Print Log, number 4 on the keypad Press number 3 on the keypad to select Usage History The system will print the serial number and the software version of the instrument the date and time, and the number of tests performed by the analyzer over the last 30 days and since the installation Press the softkey located next to Previous Screen twice to return to the System Ready display Closure time (CT) results for normal patient samples should fall within the normal reference range established for your laboratory. For example, the following chart displays CT reference ranges for a study of 176 healthy individuals with no previous history or laboratory results indicative of platelet dysfunction induced by intrinsic platelet defects, von Willebrand disease, or exposure to platelet inhibiting agents, such as Aspirin®. Samples were collected in 3.8% (0.129 M) buffered sodium citrate and tested in duplicate determinations. Samples collected in 3.2% (0.105 M) buffered sodium citrate can yield up to 12% shorter closure times than those collected using 3.8% (0.129 M) buffered sodium citrate. Differences in technique, equipment, reagents, or donor population can yield varying closure time results.   Cartridge Type Mean CT CT Reference Range Collagen/ Epinephrine 132 sec 94-193 sec Collagen/ADP 92 sec 71-118 sec PFA-100® closure time (CT) results provide an indication of platelet function. After performing a PFA-100® test, compare the CT results to the CT normal reference range established for your laboratory. Are the results for one or both test cartridges normal or abnormal? What does the test pattern indicate? To interpret CT results, use the following chart as a guide, which shows that the PFA-100® test allows you to differentiate between platelet dysfunction due to a platelet inhibiting factor (such as Aspirin® or other aspirin-containing medication) versus platelet dysfunction due to a platelet disorder. CT results above the laboratory establish cut-off range may indicate the need for further diagnostic testing. CT results should always be evaluated in conjunction with clinical history and other laboratory finding, such as bleeding time and platelet aggregometry. If CT results do not agree with the clinical assessment, additional tests should be performed.   Cartridge Type Normal Patient Aspirin® Ingestion von Willebrand Disease Glanzmann thrombasthenia   (n=176) (n=120) (n=28) (n=4) Collagen/ Epinephrine Normal Abnormal Abnormal Abnormal Collagen/ADP Normal Normal Abnormal Abnormal Several factors and medications can affect PFA-100® closure time (CT) results.  Test Limitations and Interferences Learn about test limitations and interferences. Tab TitleTextPlatelet CountIn vivo platelet plug formation requires normal functioning platelets as well as adequate numbers of platelets. The same requirements are true to obtain normal CT results in the PFA-100® test system. For this reason, patients with low platelet counts <150,000/mL (thrombocytopenia) can yield prolonged CT results that are higher than the laboratory’s established cut-off value. Note: Correlations between PFA-100® CT results and specimens with high platelet counts >500,000/mL have not been evaluated.Sedimentation RateBlood samples with high sedimentation rates may experience settling in position B while waiting to be tested in sequence with position A. If settling occurs, the hemodynamic properties of the sample may be altered, potentially affecting CT results. For this reason, it is recommended that you run samples that exhibit high sedimentation properties as single tests (using only position A). To obtain duplicate CT measurements, run a single test in position A two times. Note: In a clinical setting, it may not always be possible to judge the sedimentation properties of an unknown sample. However, the origin of the sample (which department or floor it came from), diagnosis (e.g., disease state, presence of substantial hemodilution due to cardiopulmonary bypass or ECMO, etc.), and other available information (e.g., hematocrit values) may assist you in determining if sedimentation of the sample should be considered when performing PFA-100Ò tests.HematocritSeveral clinical studies have indicated an association between significant bleeding and anemia. The mechanism may be due the inability of red blood cells to push platelets toward the periphery of a vessel to form an effective platelet plug. Because platelet plug formation is the mechanism measured by the PFA-100®, patients with low hematocrit levels <35% can also yield prolonged CT results that are higher than the laboratory’s established cut-off value. Note: Correlations between PFA-100® CT results and specimens with high hematocrit levels >50% have not been evaluated.ASAIngestion of acetyl salicylic acid (ASA), such as Aspirin®, affects platelet aggregation for up to eight days¹ by inhibiting the platelet-release reaction, which can lead to prolonged bleeding in some patients. The PFA-100® test detects ASA-induced platelet dysfunction. For example, studies have shown that 2 to 30 hours following ingestion of 325 mg of Aspirin®, 95% of normal patients will yield abnormal CTs using COL/EPI test cartridges. Of this group, 72 % will yield normal CTs using COL/ADP test cartridges. These studies show that the PFA-100® test system allows physicians to differentiate between ASA-induced platelet dysfunction versus platelet dysfunction due to platelet disorders, which could yield abnormal CTs for BOTH test cartridges. ¹ Wintrobe's Clinical Hematology, 10th Edition, (G.R. Lee, J. Foerster, J. Lukens, F. Paraskevas, J.P. Greer and G.M. Rodgers, eds), Williams & Wilkins, Baltimore, Chapter 70: 1807.MicrothrombiMicrothrombi in the sample or particulates introduced into the sample from the environment could adversely affect the PFA-100® test results and/or cause a cancellation of the test by the instrument due to the detection of a flow obstruction.Fatty Acids & LipidsCertain fatty acids and lipids found in various human diets are known to inhibit platelet function. Physicians may wish to advise patients to refrain from fatty foods prior to testing. Note: Neutral lipids, such as cholesterol, generally have no effect on platelet function.HemolysisHemolyzed blood, free hemoglobin from red blood cell lysis, can interfere with PFA-100® test results by decreasing the hematocrit and by releasing ADP (which can activate platelets).Other MedsMany medications are known to affect platelet function. Be sure to review the medication history of the patient when evaluating CT results.  To obtain accurate closure time results, collect blood samples without causing any trauma to red blood cells and platelets that could accidentally initiate the clotting process, such as platelet activation or the formation of microthrombi. To ensure closure time results for patient and control samples correlate with established normal reference range, use a consistent sample collection method, both sodium citrate concentration and venipuncture method, for drawing and handling reference range samples, patient samples, and donor control samples. Sample Collection Requirements Learn about sample collection requirements. Use a 21-gauge or larger needle to perform the venipuncture, and draw blood directly into an evacuated plastic or siliconized glass tube or syringe containing 3.2% (0.105M) or 3.8% (0.129M) buffered sodium citrate (1-part anticoagulant to 9-parts blood). A 23-gauge needle may be used for pediatric sample collection. Discard the sample if hemolyzed or if venous collapse or stoppage of blood flow occurs during collection. Gently invert the tube by hand 3 to 4 times to ensure proper mixing with anticoagulant. DO NOT place tubes on a rocker or rotator while waiting to be tested. Store samples undisturbed at room temperature. Samples are stable for up to 4 hours. DO NOT refrigerate or centrifuge samples. For the Collagen/Epinephrine (COL/EPI) test cartridge, it is recommended that testing NOT be performed until 10 minutes after blood collection to allow platelets to stabilize and reduce test variability inherent within the first 10 minutes post-collection.