INTRODUCTION — Platelets play a key role in hemostasis. Platelet dysfunction can cause a bleeding phenotype that may require specialist evaluation and interventions, especially during bleeding challenges (surgery, pregnancy, and trauma).
This topic summarizes available platelet function tests and provides information about how to interpret the results of platelet function testing.
Separate topics discuss a general approach to evaluating unexplained bleeding and the evaluation and management of specific platelet function disorders:
•Child – (See "Approach to the child with bleeding symptoms".)
•Adult – (See "Approach to the adult with a suspected bleeding disorder".)
●Platelet disorders – (See "Inherited platelet function disorders (IPFDs)".)
Platelet function testing does not have an established role in individuals with arterial thrombosis or those receiving antiplatelet therapy. Response to antiplatelet therapy is generally evaluated clinically. (See "Nonresponse and resistance to aspirin" and "Clopidogrel resistance and clopidogrel treatment failure".)
When to order platelet function testing — Generally, platelet function testing is indicated for an individual with a bleeding phenotype who has an unrevealing initial evaluation for more common bleeding disorders or bleeding out of proportion to mild abnormalities detected on initial laboratory evaluation (algorithm 1).
Indications for platelet function testing include:
●Unexplained bleeding with negative initial evaluation.
●Positive family history of a platelet function disorder.
●Genetic test results that suggest a platelet function disorder.
The initial evaluation that typically precedes platelet function testing includes:
●Thorough history of bleeding challenges, using a bleeding assessment tool (BAT), and family history of bleeding disorders.
●Complete blood count (CBC) with platelet count and examination of the peripheral blood smear.
●Standard coagulation testing with prothrombin time (PT) and activated partial thromboplastin time (aPTT).
●Screening tests for von Willebrand disease (may be omitted in some cases).
Details of the initial evaluation are presented separately:
●General evaluation – (See "Approach to the adult with a suspected bleeding disorder".)
●Platelet count – (See "Automated complete blood count (CBC)", section on 'Platelet parameters'.)
Platelet function testing generally is not indicated to evaluate abnormal platelet morphology unless the individual is also under investigation for a bleeding disorder. However, morphology review and manual platelet count assessment on a peripheral blood smear is often assessed as an initial screen for possible platelet disorders, and, when abnormal, can suggest a platelet function disorder. Morphology review is rarely sufficient as the confirmatory diagnostic test for platelet disorders, since more definitive tests are available. Several studies have demonstrated that assessment of platelet size on the blood smear is biased. (See "Evaluation of the peripheral blood smear", section on 'Platelets'.)
Platelet function testing is generally not appropriate as a means of monitoring antiplatelet drugs or of predicting bleeding in patients with thrombocytopenia.
Who should order testing — Platelet function testing is rarely available in the community. This testing is mostly available in academic medical centers and is typically ordered by a hemostasis and thrombosis expert who has access to this testing and can determine whether testing is appropriate, which testing should be done, and in what order. These experts would normally see the patients to clinically assess their bleeding phenotype prior to requesting testing.
However, in rare cases, the community physician may order platelet function testing, and/or they may receive the results of the testing and may be asked to help interpret the results for the patient and/or other consultants.
Which test(s) to order — Following initial testing of hemostasis that suggests a platelet function disorder, platelet aggregometry is the gold standard test, and, when available, this is the most useful test for a patient with a suspected platelet function disorder. (See 'Platelet aggregometry' below.)
If aggregometry is not available, other platelet function tests are sometimes available that can be obtained more rapidly and may be easier to access and easier to use but that do not always have as well validated performance characteristics. These tests may be helpful in some cases, but in other cases it may be necessary to perform aggregometry, either for diagnostic confirmation or if results of these other tests are discordant with the clinical picture. (See 'Other testing if aggregometry is not available' below.)
Genetic testing may be appropriate in many cases, especially if a heritable platelet disorder is under consideration. This generally occurs after specialist review and platelet function testing, although some individuals may be notified of pathogenic variant in a gene involved in platelet function when they have genetic testing for another reason. (See 'Evolving role of genetic testing' below.)
Measurement of bleeding time is not recommended. Sophisticated imaging such as electron microscopy may be helpful for selected cases or for research. (See 'Tests not commonly used' below.)
Caveats with testing — The following caveats may apply to test selection and interpretation:
●Medications – Some medications interfere with platelet function and will cause falsely abnormal results on platelet function testing. Prior to platelet function testing, a patient will be asked about their medication history, including any antiplatelet agents, nonsteroidal anti-inflammatory drugs (NSAIDs), and selective serotonin receptor inhibitors (SSRIs), all of which interfere with platelet function. If clinically safe, these medications should be omitted for at least a week prior to platelet function testing .
●Thrombocytopenia – Clinicians should be aware that platelet function testing can become unreliable in individuals with thrombocytopenia. There is not a strict platelet count cutoff below which this occurs. We generally avoid most of the platelet function tests (especially aggregometry) in individuals with a platelet count <80,000 microL.
However, some platelet function disorders are associated with thrombocytopenia, and laboratory assessment may be appropriate. In these cases, a method of platelet function testing independent of platelet count can be used, such as a flow cytometry-based method. (See 'Flow cytometry' below.)
●Coagulation abnormalities – The coagulation cascade that produces the fibrin mesh in a clot is carried out by soluble factors in the circulation. For tests that assay hemostasis globally, clotting abnormalities may interfere with interpretation.
●Sample handling – Platelet aggregation studies are usually performed on fresh blood samples, ideally obtained without a tourniquet . If needed, a tourniquet can be used initially and then removed after venipuncture (during blood collection). The needle size should be a 21 gauge or larger. Hemolyzed, clotted, or incompletely filled tubes should be rejected. Excessive agitation of the sample should be avoided. The sample should be maintained at room temperature (not refrigerated) and transported to the specialty testing laboratory without delay.
Uses of aggregometry — Platelet aggregometry is the gold standard test for diagnosing platelet function disorders. It is generally the preferred test when a platelet function disorder is suspected (algorithm 1).
Aggregometry measures platelet response (aggregation) to a panel of agonists:
●Adenosine diphosphate (ADP)
Collagen, thrombin, ADP, and epinephrine are physiologic platelet activators. (See "Overview of hemostasis", section on 'Platelet activation'.)
Ristocetin is an antibiotic that was withdrawn from the market because it causes platelets to agglutinate. It is used in several different tests, including aggregometry, ristocetin-induced platelet activation (RIPA), and ristocetin cofactor activity of von Willebrand factor (VWF:RCo). RIPA and VWF:RCo are used to diagnose von Willebrand disease. (See "Clinical presentation and diagnosis of von Willebrand disease", section on 'Platelet-dependent VWF activity (VWF:RCo or VWF:GPIbM)'.)
A second-line panel of agonists can sometimes be used to further investigate abnormalities:
●U46619 (a thromboxane receptor agonist)
●TRAP (thrombin receptor activating peptides) that stimulate PAR-1 (peptide sequence SFLRRN) or PAR-4 (peptide sequence AYPGKF)
●Collagen Related Peptide (CRP) and Convulxin (a rattlesnake toxin), which stimulate platelet GpVI
●Calcium ionophore A23187
Aggregometry can be performed in 96 well plates using measurements within standard plate readers . This approach allows for the assessment of multiple concentrations of agonists, since the volume of platelets required for each test is significantly lower than with aggregometry. Agonists can be lyophilized to improve standardization, and test results can be analyzed rapidly using automated spreadsheets. Fully automated aggregometry is also available .
Aggregometry can be measured on platelet rich plasma (PRP) or whole blood.
●PRP – Standard aggregometry is measured on PRP, which is obtained by centrifuging anticoagulated blood at enough force to remove red blood cells (RBCs) but not platelets (170 to 200 times gravity for 10 to 15 minutes). The PRP is stirred in a cuvette at 37°C, the agonist is added, and light transmission through the sample is measured. Platelet aggregation (first as microaggregates and later as macroaggregates) decreases the turbidity of the sample and increases light transmission. A range of agonists can be tested, each at a range of concentrations.
●Whole blood – Aggregometry on whole blood can be measured using electrical impedance with the Chronolog or Multiplate analyzer . The blood is stirred in a cuvette at 37°C, and platelets adhere to the electrode . Addition of agonists causes aggregation of additional platelets onto the electrode, which changes the impedance. The multiplate analyzer was demonstrated to be of value in screening for severe heritable platelet disorders but has lower sensitivity for milder disorders . The multiplate analyzer has also been used as a point-of-care test and for monitoring antiplatelet therapy with standardized commercial reagents.
The VerifyNow test is a type of whole blood aggregometry that has been automated. (See 'VerifyNow' below.)
The interpretation of platelet aggregometry depends on the specific pattern of aggregation to different agonists [1,6]. The figure (figure 1) shows typical aggregometry tracings for platelets from patients with a storage pool defect, Bernard-Soulier syndrome (BSS), and Glanzmann thrombasthenia (GT). The table (table 1) summarizes the typical findings with these disorders in response to different agonists.
The classic distinction between BSS and GT is as follows:
●In BSS (defect in platelet GPIb complex), there is no agglutination in response to ristocetin. Response to other agonists is normal.
●In GT (defect in platelet GPIIb-IIIa complex), there is no response to ADP, epinephrine, thrombin, or collagen. Agglutination occurs in response to ristocetin.
In some granule disorders, the second wave of aggregation may be absent with weaker platelet agonists.
Aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) cause inhibition of cyclooxygenase and a poor response to arachidonic acid stimulation. Drugs that inhibit GPIIb-IIIa (eg, abciximab) can produce a similar pattern as GT, but testing suggests that the aggregometry results underestimate the severity of the platelet function defect . Although there was initial interest in and much research on using aggregometry to monitor or assess response to antiplatelet therapies, this indication is not recommended. (See "Nonresponse and resistance to aspirin" and "Clopidogrel resistance and clopidogrel treatment failure".)
Caveats with aggregometry — Aggregometry may be inaccurate in individuals with moderate to severe thrombocytopenia (platelet count <80,000/microL). The platelet count is higher in PRP than in blood (acceptable range, 100,000 to 600,000/microL). (See 'Caveats with testing' above.)
Aggregometry is labor intensive and requires careful quality control and a fair degree of technical expertise in its performance and interpretation. Although modern aggregometers with multichannel capability, computer control, and the ability to measure adenosine triphosphate (ATP) release via luminescence have improved the technology, the test is currently limited to specialized laboratories and is not well standardized [8-10].
Aggregometry may not be sensitive to all dense granule storage pool and release defects. Although genetic testing may help to establish a diagnosis, total platelet adenine nucleotides and their release have long been measured as a more specific test for these abnormalities. Real time incorporation of ATP release into platelet aggregometry (lumi-aggregometry) enables assessment of nucleotide release simultaneously with aggregation. Alternatively, the ATP and ADP levels can be measured in platelet lysates by luminometry. (See "Overview of hemostasis", section on 'Platelet secretion' and "Inherited platelet function disorders (IPFDs)", section on 'Specific disorders'.)
A number of efforts have attempted to improve standardization, including a 2015 guideline from the International Society on Thrombosis and Haemostasis (ISTH) .
VISCOELASTIC TESTING (TEG AND ROTEM) — Viscoelastic testing is often used to confirm that hemostasis is preserved, or to guide transfusions in individuals with global hemostatic disfunction such as during major surgery or trauma. This testing can detect abnormalities of all the phases of clotting, but they cannot distinguish between thrombocytopenia and platelet function defects.
Viscoelastic testing (referred to as a global hemostasis assay) is often performed on whole blood at the patient bedside or in the operating room. The tests assay the entire hemostatic process, from platelet activation and the coagulation cascade to clot formation and lysis. Rather than measuring a single endpoint such as a clotting time, they create a tracing that illustrates the different phases of clotting over time; the different components of the tracing indicate different phases of the process (platelet function, clotting, and fibrinolysis).
There are two main viscoelastic tests:
●Thromboelastography (TEG) – TEG is the most commonly used viscoelastic test. In the TEG and the related rapid TEG (r-TEG) devices, physical properties of the clot are measured by use of a cylindrical cup that holds a whole blood sample at 37ºC and is oscillated to and fro with a rotation cycle of 10 seconds. As the clot forms, the torque of the rotating cup is transmitted to an immersed pin. The degree of pin rotation is converted to an electrical signal via a transducer and monitored via a chart recorder. The strength of the developing clot increases the magnitude of the output, whereas during clot lysis, the bonds between the cup and the pin are broken, and the signal decreases. The forces that are generated are used to measure the clotting time, kinetics of clot initiation, clot strength, and clot lysis over time (table 2).
The portions of the TEG tracing that assess platelet function are the alpha angle and the maximal amplitude, as illustrated in the figure (figure 2).
There are several devices available commercially to measure TEG. While the specifics of the devices vary, they all measure the same general parameters. TEG instruments may be insensitive to samples from patients who have taken aspirin [12-14]. However, the TEG PlateletMapping assay can overcome the limitations of thrombin-generated viscoelastic testing to reliably and accurately measure the ability of platelets to participate in clot formation and therefore measure the effects of antiplatelet drugs .
●Rotational thromboelastography (ROTEM) – The ROTEM device is an adaptation of the TEG in which the cup remains stationary and the pin rotates directly in the sample. Results obtained are essentially identical to the TEG.
The usefulness of these tests in general hematology practice remains uncertain. Indications for and uses of this testing are discussed in a 2018 Guideline from the British Society of Haematology ; more detailed information about these indications is presented separately:
•General – (See "Intraoperative transfusion and administration of clotting factors", section on 'Use of a transfusion algorithm or guideline' and "Intraoperative transfusion and administration of clotting factors", section on 'Point-of-care tests'.)
•Liver transplant – (See "Liver transplantation: Anesthetic management", section on 'Management of coagulopathy' and "Anesthesia for the patient with liver disease", section on 'Coagulation management'.)
●Postpartum hemorrhage – (See "Postpartum hemorrhage: Medical and minimally invasive management", section on 'Viscoelastic testing'.)
OTHER TESTING IF AGGREGOMETRY IS NOT AVAILABLE — Various other platelet function tests may be more readily available than aggregometry and may sometimes be obtained (table 3). The best validated is the platelet function analyzer (PFA)-100 . However, these tests are less useful than aggregometry. It is important that clinicians understand the limitations of this testing.
PFA-100 — The PFA-100 or PFA-200 is a relatively automated test performed on whole blood that simulates in vivo platelet function. The test monitors a drop in flow rate as platelets form a hemostatic plug within a small aperture (150 microns) in a membrane coated with platelet agonists (collagen, epinephrine, or ADP) [18-20]. The result is a parameter called the closure time (CT); normal ranges are presented separately. (See "Laboratory test reference ranges in adults", section on 'Platelet function analysis (PFA-100)'.)
The PFA-100 was originally validated using plasma from patients with von Willebrand disease (VWD), but it is not used to diagnose VWD because other testing is available with better performance characteristics that allows determination of the VWD subtype. (See "Clinical presentation and diagnosis of von Willebrand disease".)
The main use of the PFA-100 is as a general screening test for platelet function when a specific diagnosis is not under consideration. In an individual with a positive bleeding history who does not have access to platelet aggregometry, the PFA-100 can be used, and, if abnormal, aggregometry can be used to confirm the diagnosis of a platelet function disorder and determine the likely diagnosis (Glanzmann thrombasthenia versus Bernard-Soulier syndrome versus another disorder). (See 'Uses of aggregometry' above.)
Advantages of the PFA-100 are its speed, ease of use, and lack of requirement for special training. The test only requires 0.8 mL of citrated whole blood for each cartridge, making it attractive for pediatric samples. Normal test results can exclude severe platelet defects but not milder disorders.
The PFA is affected by a number of variables including thrombocytopenia, anemia, and low von Willebrand factor (VWF) levels . Other disadvantages include lack of specificity for a specific platelet function disorder and lack of sensitivity for and specificity for several disorders including primary secretion defects and storage pool disease.
VerifyNow — The VerifyNow test is a modified aggregometry test that uses fibrinogen-coated beads that will agglutinate in whole blood in response to various platelet activators . (See 'Platelet aggregometry' above.)
Flow cytometry — Whole blood flow cytometry is a very powerful laboratory technique for assessing platelet activation and function [22,23].
Flow cytometry can be performed on whole blood to assay platelet activation markers. It is one of the few techniques that is not adversely affected by thrombocytopenia. The following types of assays can be done:
●Assay for activation markers after exposure to platelet agonists.
●Assay platelet surface glycoprotein deficiencies seen in heritable platelet disorders (GPIIb-IIIa in Glanzmann thrombasthenia; GPIb complex in Bernard-Soulier syndrome) [24-26].
●Assay for dense granule deficiency and storage pool disease using a granule binding dye, mepacrine, in conjunction with platelet activation [27,28].
EVOLVING ROLE OF GENETIC TESTING — The role of genetic testing in platelet function disorders is evolving. Greater accessibility of genetic testing, including multigene panels, may result in genetic testing being done before (or concurrently with) platelet function testing. Information about specific genetic variants and the platelet function disorders they cause is presented separately. (See "Inherited platelet function disorders (IPFDs)", section on 'Specific disorders'.)
TESTS NOT COMMONLY USED — For the majority of the 20th century, the only means of assessing platelet function were a small number of fairly unreliable tests (manual platelet count, inspection of the peripheral blood smear, bleeding time).
The following tests are not routinely used in the evaluation of platelet function disorders, although they may have a limited role in selected circumstances.
●Bleeding time – The bleeding time is rarely used and is mostly of historical interest. The test was invented in 1910 and was performed through the 1980s and perhaps beyond, but it has been supplanted by other testing . It involves using a device to make a small cut on the patient's forearm and wiping away the blood without dislodging the clot until bleeding ceases. The test requires specialized training and is highly operator-dependent and time-consuming, and studies suggest it is not a good test to predict surgical bleeding . It may leave a scar in some individuals.
●Electron microscopy (EM) – EM can be extremely informative for certain disorders such as abnormalities of platelet granules. (See "Inherited platelet function disorders (IPFDs)", section on 'Specific disorders'.)
However, it is labor-intensive and generally reserved for unusual cases or research studies.
●Less validated tests of hemostasis – A number of specialized tests have been developed that may be useful for research but have not been as well validated in clinical practice as aggregometry and other tests listed above. (See 'Platelet aggregometry' above and 'Other testing if aggregometry is not available' above.)
•Clot signature analyzer – The clot signature analyzer (CSA) is a test of global hemostasis that measures formation of a hemostatic plug in a tube with flowing blood. Platelets are activated by exposure to collagen in the tube or by punching a hole in the tube [31,32].
•Thrombotic status analyzer – The thrombotic status analyzer (TSA) measures platelet activation in whole blood in a capillary tube by assaying occlusion of the tube .
•Cone and plate analyzer – The cone and plate(let) analyzer measures adhesion of platelets to extracellular matrix (ECM) under flow conditions using whole blood [34-36]. Platelet adhesion and aggregation on the surface of the plate are monitored by an image analyzer. Adhesion and aggregation require platelet GPIIb-IIIa, GPIb, and plasma von Willebrand factor (VWF). The test displays two parameters: surface coverage (SC) and average size (AS). The SC is a measure of the percentage of the plate covered by platelets, and the AS is the means size of surface bound objects
The main advantages of this system are that it uses a small volume of blood (150 to 250 microL), yields results in 5 to 15 minutes, and exhibits a close resemblance to in vivo physiologic conditions.
•Microfluidic devices – Researchers have developed a number of microfluidic devices that measure real time thrombus formation in whole blood. The main use of these devices is for research, but some approaches to investigate platelet function under flow within microfluidic devices appear promising in clinical settings . One example is the Total Thrombus formation analysis system (T-TAS) . This a flow-microchip chamber with thrombogenic surfaces that generates images of thrombi in real time that imitate vessel wall injury and produce quantitative data. T-TAS can be used for monitoring antithrombotic therapy and investigating patients with suspected platelet function defects.
•ICHOR POC Hematology Counter – When platelets within anticoagulated whole blood samples are stimulated by agonists (eg, ADP, epinephrine, ristocetin), they form aggregates, with a resulting reduction in the number of free platelets. A point-of-care (POC) assay that uses counting of the remaining platelets allows counting of platelets before and after agonist stimulation has been developed (ICHOR Point of Care Hematology Counter) [39,40].
•Hemostatus device – The Hemostatus device is a POC device that measures platelet-mediated thrombin generation, a simple physiologic assay of platelet function. The activator is platelet activating factor and the assay is based on the kaolin-activated clotting time [41,42]. The test is insensitive to aspirin and GPIb function, but it can detect abnormalities in GPIIb-IIIa.
•Hemodyne – The Hemodyne instrument assays global platelet function by measuring forces generated during clotting . Platelet contractile force (PCF) is a test of global platelet function. It measures platelet activation by assaying clot retraction, which occurs following clot formation. Blood or platelet rich plasma is placed within a small cup between two parallel plates and treated with thrombin or other agonist. Retraction (size reduction) depends on initial activation with shape change and pseudopod formation, followed by adherence of pseudopods to a fibrin mesh (mediated by receptors such as GPIIb-IIIa). The retraction force is detected by a transducer. Two measurements are made :
-Platelet contractile force (PCF) – The force activated platelets exert on the fibrin network; this is abnormally low in thrombocytopenia, platelet function disorders, and with GPIIb-IIIa inhibitors [45,46].
-Elastic modulus (EM) – The rigidity of the clot; this is abnormally low in hypofibrinogenemia, clotting factor deficiencies, and with anticoagulants.
●Soluble activation markers – In the research setting, platelet activation has been assayed using measurement of products released from activated platelets. In these methods, blood samples are usually collected into tubes containing inhibitors of platelet activation (theophylline and PGE1) and platelet-specific proteins are assayed. Examples of these proteins include platelet factor 4 (PF4) and thromboglobulin, which are released from alpha granules. The ratio of these two proteins often indicates if there has been a problem with artifactually induced activation.
Other proteins assayed have included plasma soluble P-selectin, soluble CD40 ligand, glycoproteins V and VI, and thromboxane B2 (a metabolite of thromboxane A2), or, in urine, the thromboxane metabolite 11-dehydrothromboxane B2 . P-selectin is an alpha granule marker that can also be assayed on the platelet surface by flow cytometry. (See 'Flow cytometry' above.)
Uptake and release of radiolabeled serotonin from the dense granules was used in the past to assess storage and release defects. These assays have a number of disadvantages, including false elevation of levels due to sample handling, insensitivity, need for radioactive materials, and high cost.
SUMMARY AND RECOMMENDATIONS
●Indications – Platelet function testing is appropriate for unexplained bleeding with negative initial evaluation, or for bleeding out of proportion to mild abnormalities on laboratory testing (algorithm 1). It may also be indicated when there is a known platelet function disorder in a first-degree relative, or genetic test results suggestive of a platelet function disorder. Platelet function testing is generally not appropriate as a means of monitoring antiplatelet drugs or for predicting bleeding in patients with thrombocytopenia. Testing is mostly available in academic medical centers and generally ordered by hemostasis and thrombosis experts. (See 'When to order platelet function testing' above and 'Who should order testing' above.)
●Choice of test – Platelet aggregometry is the gold standard test, and, when available, this is the most useful test for a patient with a suspected platelet function disorder. Testing may be inaccurate if the patient is taking an antiplatelet medication or has thrombocytopenia (platelet count <80,000/microL). (See 'Which test(s) to order' above and 'Caveats with testing' above.)
●Aggregometry – Aggregometry measures platelet response (aggregation) to a panel of agonists (collagen, ADP, ristocetin) (figure 1). It can be performed on platelet rich plasma or whole blood. Interpretation depends on the pattern of response to specific agonists. The table (table 1) summarizes the aggregometry findings in common disorders.
●TEG – Thromboelastography (TEG) and rotational thromboelastography (ROTEM) are point-of-care global hemostasis tests (also called viscoelastic tests) that assay platelet function, fibrin formation (coagulation factor function), and fibrinolysis (clot dissolution) (figure 2). The principle involves measuring the torque between a cup and pin as a clot forms and prevents rotation of one of the pieces (in TEG, the cup rotates; in ROTEM, the pin rotates). The main use of these tests is to guide transfusion during surgery, in trauma, and with postpartum hemorrhage. TEG and ROTEM cannot distinguish platelet dysfunction from thrombocytopenia. (See 'Viscoelastic testing (TEG and ROTEM)' above.)
●Genetic testing – Genetic testing has an increasing role in diagnosing platelet function disorders. (See 'Evolving role of genetic testing' above.)
●Tests reserved for research or unusual cases – The bleeding time is of historical interest and is not indicated in investigation of bleeding disorders. Morphology review may be helpful but is prone to observer bias and is rarely sufficient to make a specific diagnosis. Electron microscopy is labor intensive and often reserved for unusual cases or research. (See 'Tests not commonly used' above.)
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