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External Quality Assurance of Platelet Function Assays: Results of the College of American Pathologists Proficiency Testing Program.

Platelets perform multiple functions related to hemostasis and can be activated by multiple pathways; thus, no single platelet function test can assess all aspects of platelet function or diagnose all the different abnormalities that can occur. Platelet function assays are used to screen for and diagnose hereditary and acquired platelet function disorders as well as to screen for von Willebrand disease. (1, 2) In addition, platelet function assays are used to monitor antiplatelet medications including clearance of antiplatelet agents before surgery. (3-5) Owing to platelet function assay design differences, the results between assays are often not comparable. (6) Regardless of the use or design of the assay, quality assurance is important for all methods that are intended for use clinically. Quality assurance includes both internal quality assurance, such as quality control, and external quality assurance (EQA)/proficiency testing. Proficiency testing consists of sending unknown samples to laboratories performing clinical testing. Laboratories test the samples and return the results to the EQA organization, which evaluates the results and provides feedback to participating laboratories on their results versus those of other laboratories and on whether they passed the proficiency test. Proficiency testing is required in the United States and many other countries for laboratories to be certified to perform clinical testing. The College of American Pathologists (CAP) is the largest source of proficiency testing in the United States.

For most hemostasis proficiency tests, a lyophilized plasma or serum sample is prepared and sent to participating laboratories for testing. This type of EQA sample is impossible for platelet function testing, which requires fresh blood containing functional platelets with limited stability. Lack of EQA programs has been cited as a problem for overall quality assurance related to platelet function testing. (7) In 2006, the CAP Coagulation Resource Committee first offered an EQA program that combined testing for the Platelet Function Analyzer PFA-100 and platelet aggregation. The CAP platelet function EQA program was briefly described in 2007. (8) The CAP approach is to have laboratories collect normal citrated anticoagulated whole blood that is added to challenge tubes containing either saline, to simulate a normal sample, or tirofiban, a GPIIbIIIa (integrin [[alpha].sub.IIb][[beta].sub.3]) inhibitor that blocks platelet aggregation, to simulate an abnormal sample, followed by platelet function testing. Unlike assays that measure a single analyte (like sodium), these platelet function tests measure different aspects of platelet function. Tirofiban was chosen as a simulated abnormality that could be detected in all of the assays.

A similar approach for the PFA-100 was developed by the Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) in 2009 and updated in 2014. (9, 10) The program's approach was to obtain a sample and make a baseline measurement, followed by addition of the normal whole blood to challenge tubes designed to mimic normal, mild to moderate, and severe defects. The RCPAQAP provided no information on what was added to the tubes to produce the platelet function abnormalities, making an assessment of what type of defects these might simulate difficult. (11) The CAP platelet function EQA program was expanded to include proficiency testing for optical (platelet-rich plasma) platelet aggregation, impedance platelet aggregation, PFA-100, PlateletWorks, and PlateletMapping. This report summarizes the results of platelet function proficiency testing for these 5 methods for 9 surveys from 2012 to 2016.

MATERIALS AND METHODS

Sample Preparation and Testing

At present, there is no way to stabilize active platelets in large amounts of whole blood for shipping to thousands of clinical laboratories in the same way that large lyophilized pools of plasma can be prepared for other types of proficiency testing. For the platelet function assays in this study, namely, optical platelet aggregation, impedance platelet aggregation, PFA-100 testing, PlateletWorks platelet aggregation, and PlateletMapping, proficiency testing required each participating clinical laboratory to obtain blood from a normal donor that was anticoagulated with 3.2% sodium citrate. The normal donor must not have taken aspirin or any other antiplatelet drug in the prior 2 weeks. Two specimen challenge tubes were sent with each platelet function survey. One tube contained saline, while the other contained tirofiban. If a normal donor was used, the saline challenge should provide normal results. Tubes were obtained from Haematologic Technologies Inc (Essex Junction, Vermont). Tirofiban simulates a severe platelet aggregation defect similar to homozygous GPIIbIIIa deficiency (Glanzmann thrombasthenia). Tirofiban was expected to produce an abnormal result for all types of platelet function testing. The participating clinical laboratory was instructed to pipette citrated whole blood from the normal donor into each of the challenge tubes and mix gently by inversion 8 to 10 times. Platelet function testing was then performed on these samples according to the standard procedure for each laboratory.

Platelet Aggregation Testing.--Proficiency testing was provided for 2 types of platelet aggregation testing: optical aggregometry and impedance aggregometry. In optical aggregometry, platelet aggregation is measured by using light transmission through platelet-rich plasma. In impedance aggregometry, aggregating platelets bind to electrodes in the sample, increasing resistance or impedance. An impedance versus time curve is measured and maximum impedance reported.

To prepare samples for testing, 6 mL of 3.2% citrated anticoagulated whole blood was added to tubes containing saline or tirofiban. In the 2012 and 2013 surveys the final tirofiban concentration was 70 ng/mL whole blood. This produced very low average percentage aggregation. The CAP Coagulation Resource Committee decided to change the concentration of tirofiban to 35 ng/mL in 2014 to better simulate a clinical platelet aggregation defect. For optical platelet aggregation, laboratories were instructed to prepare platelet-rich plasma according to their laboratory procedure. Separate adenosine diphosphate (ADP) and epinephrine agonist reagents were supplied with the survey kit. The final concentrations of agonist in the test cuvette were 10 iM for ADP and 10 iM for epinephrine. For impedance aggregation, laboratories were instructed to use the ADP reagent supplied with their assay rather than the survey agonists. Maximum aggregation or maximum impedance was recorded for each challenge.

PFA-100 Testing.--The PFA-100 (Siemens Healthcare Diagnostics, Tarrytown, New York) is an automated instrument that measures the ability of platelets to form a platelet plug. To prepare samples for testing, 4.5 mL of citrated anticoagulated whole blood was added to tubes containing saline or tirofiban (final concentration 70 ng/mL whole blood), gently mixed, and tested by using the collagen/adenosine diphosphate (COL/ADP) and collagen/epinephrine (COL/EPI) cartridges. Closure time and error messages were recorded for each challenge. An error message indicating maximum time exceeded was assigned a closure time of 300 seconds. All other error measures were not included in the data for this study.

PlateletWorks Testing.--The PlateletWorks assay (Helena Laboratories, Beaumont, Texas) measures the difference in platelet counts in the sample before and after agonist addition. An ethylenediaminetetraacetic acid (EDTA) sample was drawn from the normal donor and a platelet count determined. EDTA whole blood (6 mL) from the normal donor was added to tubes containing saline or tirofiban (final concentration 70 ng/mL), gently mixed, and tested by using the PlateletWorks-ADP and PlateletWorks-Collagen tubes. Platelet counts were performed on each sample tube and the percentage platelet aggregation was calculated from the EDTA platelet count and sample tube platelet counts. The calculated percentage aggregation was recorded for each challenge.

PlateletMapping.--PlateletMapping (Haemoscope Corporation, Niles, Illinois) is a modification of the standard thromboelastography that measures clot viscoelasticity as a measure of platelet aggregation with different agonists. Sodium heparin and sodium citrate samples were collected from normal donors. Heparinized whole blood (2.0 mL) was added to tubes containing saline or tirofiban (263 ng/mL final concentration) and gently mixed. Citrated whole blood and the 2 challenge tubes containing heparinized whole blood were tested per manufacturer instructions.

Result Evaluation and Statistical Analysis.--Platelet function proficiency testing challenges are sent to participating clinical laboratories twice each year. Proficiency testing results reported from 9 surveys were collected and analyzed in this study (2 surveys each from 2012, 2013, 2014, and 2015 and 1 survey from 2016). The mean, median, standard deviation, and interquartile range (box on box and whisker plots) were determined for results from each challenge type (saline or tirofiban) for each platelet function assay. Participants were asked to interpret the results from each challenge as normal or abnormal (or inhibited or not inhibited for PlateletMapping). The expected correct or accurate result for the test was the interpretation of normal or not inhibited for the saline sample and abnormal or inhibited for the tirofiban sample. The percentage of participants interpreting challenges as normal, abnormal, or no interpretation was determined for all saline and all tirofiban results for a given platelet function assay. The [chi square] test was used to determine if there were significant differences in the percentage of applicants reporting no interpretation among different assays and which assays had different percentages. A P value less than .05 is considered a statistically significant result.

RESULTS

Platelet Aggregation Survey, PF

In 2012, a total of 193 laboratories participated in the optical aggregation survey. The number of participating laboratories remained stable at approximately this number through 2016. For optical aggregation, the interquartile ranges for maximum aggregation on the saline challenges were 70% to 90% for both ADP and epinephrine agonists (Figure 1, A and B, and Figure 2, A and B; Table). For the tirofiban sample, most laboratories reported low maximum aggregations in the 0% to 10% range for surveys in 2012 and 2013, using a tirofiban concentration of 70 ng/mL whole blood. The tirofiban concentration was decreased to 35 ng/ mL whole blood starting in 2014, which resulted in the higher percentage maximum aggregation results of approximately 10% to 30% for ADP and 5% to 15% for epinephrine.

With either ADP or epinephrine agonist, there was good separation between the maximum aggregation results reported as normal for saline versus abnormal for tirofiban. An example of individual results for 1 survey are shown in Figure 1, A and B, and in Figure 2, A and B. Overall 7444 of 7813 survey responses (95%) provided an interpretation of platelet aggregation survey results and of those, 7015 of 7444 (94%) provided the correct interpretation (normal for saline and abnormal for tirofiban).

In 2012, a total of 43 laboratories participated in the impedance aggregation survey, and by 2016 this number increased to 65. For impedance aggregation, the interquartile ranges for maximum aggregation were 10 to 20 ohms for the saline and 0 to 2 ohms for tirofiban challenges (Figure 3, A and B). Lowering the tirofiban concentration had only a small effect on whole blood maximum aggregation, increasing average maximum aggregation from 0.1 to 0.8 ohms. There was good separation between the maximum aggregation results reported as normal for saline versus abnormal for tirofiban. Overall 965 of 1015 survey responses (95%) provided an interpretation of whole blood platelet aggregation survey results, and of those, 944 of 965 (98%) provided the correct interpretation (normal for saline and abnormal for tirofiban).

PFA-100 Survey, PF1

In 2012, a total of 1205 laboratories participated in the PFA-100 survey, and by 2016 this number increased slightly to 1225. For the PFA-100, the interquartile ranges for closure time was 75 to 100 seconds for the COL/ADP cartridge and 100 to 140 seconds for the COL/EPI cartridge for the saline sample (Figures 4, A and B; and 5, A and B). For the tirofiban sample, most laboratories reported maximum closure times of 300 seconds for both COL/ ADP and COL/EPI. In general, there was good separation between the closure time results reported as normal for saline versus abnormal for tirofiban for both cartridges. Overall 44,952 of 45,616 survey responses (99%) provided an interpretation of PFA-100 survey results, and of those, 42,934 of 44,952 (96%) provided the correct interpretation (normal for saline and abnormal for tirofiban).

PlateletWorks Survey, PF1

In 2012, a total of 65 laboratories participated in the PlateletWorks survey, and by 2016 this number increased to 85. For PlateletWorks, the interquartile ranges for percentage of aggregation on the saline sample were 90% to 99% for ADP and 70% to 95% for collagen (Figures 6, A and B; and 7, A and B). For the tirofiban sample, PlateletWorks results were more variable using collagen than for PlateletWorks using ADP or results seen in platelet aggregation and PFA-100 testing. Laboratories reported percentage aggregations of about 15% to 50% for ADP and 15% to 60% for collagen with overlap of saline versus tirofiban results. Overall 2412 of 2454 survey responses (98%) provided an interpretation of PlateletWorks survey results, and of those, 1207 of 1276 (95%) provided the correct interpretation using ADP (normal for saline and abnormal for tirofiban), but only 936 of 1136 (82%) provided the correct interpretation using collagen.

PlateletMapping Survey, PLTM

In 2012, a total of 120 laboratories participated in the PlateletMapping survey, and by 2016 this number increased to 202. For PlateletMapping, the interquartile ranges for percentage of inhibition on the saline sample were 10% to 45% for ADP and 5% to 30% for arachidonic acid (Figures 8, A and B; and 9, A and B). For the tirofiban sample, interquartile ranges for percentage inhibition were 90% to 100% for ADP, but 40% to 80% for arachidonic acid with substantial overlap between saline and tirofiban results. Unlike the other surveys where most of the participants provided an interpretation, only 2267 of 5301 survey responses (43%) provided an interpretation for the PlateletMapping results. The percentage of laboratories providing an interpretation for PlateletMapping was lower than for all the other platelet function tests evaluated ([chi square], P < .001). For those that did provide an interpretation, it was often incorrect. For ADP, 1128 of 2697 survey responses (42%) provided an interpretation, but only 927 of 1128 (82%) were correct. For arachidonic acid, 1139 of 2604 survey responses (44%) provided an interpretation and 964 of 1139 (85%) were correct. Results were worse for saline versus tirofiban challenges. On the saline challenge using ADP as the agonist, only 366 of 1352 (27%) correctly identified this as not inhibited (normal or correct response), while 195 of 1352 (14%) reported inhibited (abnormal or incorrect) and 791 of 1352 (59%) provided no interpretation. On the saline challenge using arachidonic acid as the agonist, only 469 of 1308 (36%) correctly identified this as not inhibited (normal or correct response), while 104 of 1308 (8%) reported inhibited (abnormal or incorrect) and 735 of 1308 (56%) provided no interpretation. On the tirofiban challenge overall 1056 of 2641 (40%) reported inhibited (abnormal and correct), 77 of 2641 (3%) reported not inhibited (normal and incorrect), while 1508 of 2641 (57%) provided no interpretation. There did not seem to be a clear cutoff for interpretation. Similar percentage inhibitions were reported as both inhibited and not inhibited.

DISCUSSION

In 2006, the CAP was the first to provide an EQA program using blood drawn at the participating laboratory for platelet aggregation and PFA-100, with an initial report on progress in 2007 for the PFA-100.8 In this EQA scheme, blood is drawn and subsequently added to challenge tubes, provided by the CAP, that contain either saline or tirofiban. The CAP has now extended this type of platelet function proficiency testing to platelet aggregation, PlateletWorks, and PlateletMapping in more than 1000 laboratories. Results of this program during the last 4 to 5 years have demonstrated that most clinical laboratories and most platelet function tests perform well with accuracy and precision similar to other clinical assays.

The PFA-100 assay is most often used as a screen for moderate to severe von Willebrand disease and other platelet function abnormalities. (12) It can detect antiplatelet medications in some patients, but it is typically not used to monitor this type of drug. In 2009, the RCPAQAP offered an EQA program for platelet function testing on the PFA-100 that was updated in 2014. (10) Normal blood drawn at the host laboratory was added to 4 challenge tubes intended to represent normal, aspirin-like defect, moderate defect, and severe defect. The aspirin-like challenge failed owing to stability issues. Results were presented for normal, moderate/severe, and severe defects. The RCPAQAP study reported that the PFA-100 had good reproducibility with CVs of 15% to 25% for normal, or lower for severe defects. The approach in the CAP proficiency for the PFA-100 survey was similar to the RCPAQAP but simplified to test only normal versus severe abnormality. PFA-100 reproducibility was similar in our study with CVs of 21% to 23% for the saline challenge (normal) and lower for the tirofiban challenge (severe abnormality), where most laboratories reported more than 300 seconds for closure time. Overall 44,952 of 45,616 survey responses (99%) provided an interpretation, and 42,934 of 44,952 (96%) gave the correct interpretation. By 2013 the RCPAQAP reported enrolling 50 laboratories in the PFA-100 EQA program. By 2016, CAP had enrolled more than 1200 laboratories in the PFA-100 proficiency testing survey, a substantial expansion of platelet function EQA.

Platelet aggregation is typically the test that is performed if a result from a screening test like PFA-100 or Platelet Works is positive. (12) Platelet aggregation can diagnose a variety of inherited platelet disorders including abnormalities of the GP1b-V-IX complex (Bernard Soulier syndrome), GPIIbIIIa (Glanzmann thrombasthenia), ADP receptor, thromboxane pathway, and secretion pathways. Platelet aggregation can also be used to evaluate the effect of antiplatelet medications including aspirin, clopidogrel, and other ADP receptor blockers. (3) While platelet aggregation testing, using either optical (platelet-rich plasma) or impedance (whole blood) methodology, is considered the gold standard for platelet function testing, concerns have been raised about variability in how the test is performed. (9, 13)

Our study is the first description of a proficiency test of platelet aggregation performance. The proficiency testing evaluated whether laboratories could differentiate normal from a severe platelet aggregation defect. In this study, optical and impedance platelet aggregation testing performed well on proficiency testing. For combined optical and impedance aggregation, 8409 of 8828 survey responses (95%) provided an interpretation of the results, with 7959 of 8409 (95%) giving the correct interpretation (normal for saline and abnormal for tirofiban). While separation of normal versus a severe abnormality may seem like an achievable goal, it was not achieved by all the methods evaluated with this proficiency testing program.

The PlateletWorks test is a simple maximum platelet aggregation assay based on sample platelet counts before and after addition of ADP or collagen as agonists. (14) There is limited clinical experience with this technology. It has been used to monitor aspirin, ADP receptor, and GPIIb/IIIa receptor antagonist antiplatelet drugs and to monitor platelet function after open heart surgery. (2) Clinical laboratories participating in PlateletWorks proficiency testing performed well when using ADP, with overall 1276 of 1298 survey responses (98%) providing an interpretation of the results and 1207 of 1276 (95%) giving the correct interpretation. Testing with collagen as the agonist did not perform as well in the PlateletWorks assay; 1136 of 1156 survey responses (98%) provided an interpretation, but only 936 of 1136 (82%) provided the correct interpretation.

The original thromboelastograph methodology was not sensitive to platelet-inhibiting drugs owing to the high level of platelet activation by thrombin generated in the assay. (4) A modified thromboelastograph assay termed PlateletMapping was developed that uses 4 separate tests, 2 different sample types (heparinized and citrated blood), and 3 agonists (ADP, arachidonic acid, and thrombin generated by contact activation), resulting in an estimate of the percentage of inhibition of the platelet response to ADP or arachidonic acid. PlateletMapping has been used to monitor antiplatelet therapy and to detect platelet dysfunction before surgery,2,4 with variable results. Of the platelet function assays evaluated in the CAP EQA program, PlateletMapping had the worst performance. There was substantial overlap of inhibited versus not inhibited results, leading to apparent confusion on how to interpret the results by participants as demonstrated by the fact that overall only 2267 of 5301 of survey responses (43%) provided an interpretation and of those that did provide one, many were wrong. For ADP PlateletMapping on the saline sample, only 366 of 1352 total survey responses (27%) correctly identified the sample as not inhibited (normal), while 791 of 1352 (59%) provided no interpretation and 195 of 1352 (14%) provided the wrong interpretation. For arachidonic acid, only 469 of 1308 (36%) provided a correct interpretation for the saline sample. This compares to 42,934 of 45,616 (94%) overall identifying normal for PFA-100.

Our findings of poor performance for PlateletMapping are consistent with other evaluations. (15) Recent studies have reported that compared to VerifyNow, platelet aggregation, and flow cytometry, PlateletMapping demonstrated the worst performance, poor precision, and poor reliability. (16) The authors' final conclusion was that PlateletMapping "is least suited to monitor the effect of antiplatelet agents." Others have reported serious design and operational issues, including the use of heparinized samples for platelet function analysis that lead to platelet activation in the sample, overestimation of platelet inhibition in patients not taking medications, (17) lack of utility for predicting preoperative bleeding risk, (18) postoperative bleeding, (19) detection of antiplatelet therapy in trauma patients, (20) and lack of utility for monitoring platelet inhibitor therapy. (21) The inability of participants to confidently discriminate normal platelet function in this study highlights the limited utility of PlateletMapping.

In summary, platelet aggregation (optical and impedance), PFA-100, and PlateletWorks using ADP as an agonist performed well on proficiency testing with more than 90% of laboratories providing an interpretation and a similar number giving the correct answer. PlateletWorks using collagen and PlateletMapping using either ADP or arachidonic acid showed worse accuracy than the other methods. For all of the methods except PlateletMapping, 95% to 99% of participants provided an interpretation, most of which were correct.

The platelet function proficiency testing offered by CAP is the first of its kind for platelet aggregation, PlateletWorks, and PlateletMapping. While it provides an indication of whether laboratories can differentiate normal from a severe abnormality (significant blockade of platelet aggregation), it does not test whether laboratories can correctly diagnose specific types of platelet function defects like storage pool deficiency or Bernard Soulier syndrome, among others. Testing the ability of each platelet function assay to detect specific platelet abnormalities was beyond the scope of this type of proficiency testing. Within these limitations, it was suggestive that for platelet aggregation, PFA-100, and PlateletWorks-ADP, most laboratories can at least separate normal from severe abnormal platelet activity. This was less obvious for other platelet function tests.

References

(1.) Gurney D. Platelet function testing: from routine to specialist testing. Br J Biomed Sci. 2016;73(1):10-20.

(2.) Paniccia R, Priora R, Liotta AA, Abbate R. Platelet function tests: a comparative review. Vasc Health Risk Manag. 2015;11:133-148.

(3.) Orme R, Judge HM, Storey RF. Monitoring antiplatelet therapy. Semin Thromb Hemost. 2017;43(3):311-319.

(4.) Agarwal S. Platelet function testing in cardiac surgery. Transfus Med. 2016; 26(5):319-329.

(5.) Gorog DA, Fuster V. Platelet function tests in clinical cardiology: unfulfilled expectations. I Am Coll Cardiol. 2013;61(21):2115-2129.

(6.) Berger PB, Kirchner HL, Wagner ES, et al. Does preoperative platelet function predict bleeding in patients undergoing off pump coronary artery bypass surgery? J Interv Cardiol. 2015;28(3):223-232.

(7.) Hayward CP, Eikelboom J. Platelet function testing: quality assurance. Semin Thromb Hemost. 2007;33(3):273-282.

(8.) Cunningham MT, Brandt JT, Chandler WL, et al. Quality assurance in hemostasis: the perspective from the College of American Pathologists proficiency testing program. Semin Thromb Hemost. 2007;33(3):250-258.

(9.) Favaloro EJ. Internal quality control and external quality assurance of platelet function tests. Semin Thromb Hemost. 2009;35(2):139-149.

(10.) Favaloro EJ, Bonar R. External quality assessment/proficiency testing and internal quality control for the PFA-100 and PFA-200: an update. Semin Thromb Hemost. 2014;40(2):239-253.

(11.) Favaloro EJ, Bonar R. External quality assurance for the PFA-100(R). J Thromb Haemost. 2011;9(4):878-880.

(12.) Favaloro EJ. Clinical utility of closure times using the platelet function analyzer-100/200. Am J Hematol. 2017;92(4):398-404.

(13.) Moffat KA, Ledford-Kraemer MR, Nichols WL, Hayward CP. Variability in clinical laboratory practice in testing for disorders of platelet function: results of two surveys of the North American Specialized Coagulation Laboratory Association. Thromb Haemost. 2005;93(3):549-553.

(14.) Campbell J, Ridgway H, Carville D. Plateletworks: a novel point of care platelet function screen. Mol Diagn Ther. 2008;12(4):253-258.

(15.) Chandler WL. Platelet function assays: not all are created equal. Clin Chem. 2014;60(12):1469-1470.

(16.) Karon BS, Tolan NV, Koch CD, et al. Precision and reliability of 5 platelet function tests in healthy volunteers and donors on daily antiplatelet agent therapy. Clin Chem. 2014;60(12):1524-1531.

(17.) Nelles NJ, Chandler WL. Platelet mapping assay interference due to platelet activation in heparinized samples. Am J Clin Pathol. 2014;142(3):331-338.

(18.) Alstrom U, Granath F, Oldgren J, Stahle E, Tyden H, Siegbahn A. Platelet inhibition assessed with VerifyNow, flow cytometry and PlateletMapping in patients undergoing heart surgery. Thromb Res. 2009;124(5):572-577.

(19.) Carroll RC, Chavez JJ, Snider CC, Meyer DS, Muenchen RA. Correlation of perioperative platelet function and coagulation tests with bleeding after cardiopulmonary bypass surgery. J Lab Clin Med. 2006;147(4):197-204.

(20.) Daley MJ, Trust MD, Peterson EJ, et al. Thromboelastography does not detect preinjury antiplatelet therapy in acute trauma patients. Am Surg. 2016; 82(2):175-180.

(21.) Collyer TC, Gray DJ, Sandhu R, BerridgeJ, Lyons G. Assessment of platelet inhibition secondary to clopidogrel and aspirin therapy in preoperative acute surgical patients measured by Thrombelastography Platelet Mapping. Br J Anaesth. 2009;102(4):492-498.

Wayne L. Chandler, MD; Alan F. Brown, MD; Dong Chen, MD, PhD; Karen Moser, MD; John D. Olson, MD, PhD; Huy Phu Pham, MD; Kristi J. Smock, MD; Oksana Volod, MD; Russell A. Higgins, MD

Accepted for publication July 1 8, 2018.

Published online December 21, 2018.

From the Department of Laboratories, Seattle Children's Hospital, Seattle, Washington (Dr Chandler); the Department of Pathology, University of Utah, Salt Lake City (Drs Brown and Smock); Special Coagulation Laboratory, Mayo Clinic, Rochester, Minnesota (Dr Chen); the Department of Pathology, St. Louis University School of Medicine, St Louis, Missouri (Dr Moser); the Department of Pathology, University of Texas Health Science Center, San Antonio (Drs Olson and Higgins); the Department of Pathology, University of Alabama at Birmingham (Dr Pham); and the Department of Pathology, Cedars-Sinai Medical Center, Los Angeles, California (Dr Volod).

Dr Volod is a consultant with Haemonetics Inc. The other authors have no relevant financial interest in the products or companies described in this article.

Corresponding author: Wayne L. Chandler, MD, Department of Laboratories, OC.8.720, Seattle Children's Hospital, 4800 Sandpoint Way NE, Seattle, WA 98105 (email: wayne.chandler@ seattlechildrens.org).

Caption: Figure 1. Optical aggregation using adenosine diphosphate (ADP). A, Box and whisker plots of maximum (Max) optical aggregation results for each survey separated by sample type (saline versus tirofiban). B, An example of individual laboratory responses for 1 survey.

Caption: Figure 2. Optical aggregation using epinephrine (EPI). A, Box and whisker plots of maximum (Max) optical aggregation results for each survey separated by sample type (saline versus tirofiban). B, An example of individual laboratory responses for 1 survey.

Caption: Figure 3. Impedance aggregation using adenosine diphosphate (ADP). A, Box and whisker plots of maximum (Max) impedance aggregation results for each survey separated by sample type (saline versus tirofiban). B, An example of individual laboratory responses for 1 survey.

Caption: Figure 4. Platelet Function Analyzer 100 (PFA-100) closure times using the collagen/ adenosine diphosphate (COL/ADP) cartridge. A, Box and whisker plots of closure time results in seconds for each survey separated by sample type (saline versus tirofiban). B, An example of individual laboratory responses for 1 survey.

Caption: Figure 5. Platelet Function Analyzer 100 (PFA-100) closure times using the collagen/ epinephrine (COL/FPI) cartridge. A, Box and whisker plots of closure time results in seconds (sec) for each survey separated by sample type (saline versus tirofiban). B, An example of individual laboratory responses for 1 survey.

Caption: Figure 6. PlateletWorks aggregation using adenosine diphosphate (ADP). A, Box and whisker plots of percentage aggregation results for each survey separated by sample type (saline versus tirofiban). B, An example of individual laboratory responses for 1 survey.

Caption: Figure 7. PlateletWorks aggregation using collagen (Col). A, Box and whisker plots of percentage aggregation results for each survey separated by sample type (saline versus tirofiban). B, An example of individual laboratory responses for 1 survey.

Caption: Figure 8. PlateletMapping percentage inhibition using adenosine diphosphate (ADP). A, Box and whisker plots of percentage inhibition results for each survey separated by sample type (saline versus tirofiban). B, An example of individual laboratory responses for 1 survey.

Caption: Figure 9. PlateletMapping percentage inhibition using arachidonic acid (AA). A, Box and whisker plots of percentage inhibition results for each survey separated by sample type (saline versus tirofiban). B, An example of individual laboratory responses for 1 survey.
Summary of Results

                                           Saline

Assay                      Interpretation    N      % Aggregation,
                                                    Mean [+ or -] SD

Optical aggregation,       Normal           1654     82 [+ or -] 14
  epinephrine              Abnormal          166     33 [+ or -] 25
                           No interp         109
Optical aggregation, ADP   Normal           1739     82 [+ or -] 14
                           Abnormal          156     36 [+ or -] 24
                           No interp          77

                                             N           Ohms,
                                                    Mean [+ or -] SD

Impedance aggregation,     Normal            469   16.5 [+ or -] 6.1
  ADP                      Abnormal           16   10.3 [+ or -] 10.4
                           No interp          25

                                             N       Closure sec,
                                                    Mean [+ or -] SD

PFA-100, Col/ADP           Normal          10,426    87 [+ or -] 20
                           Abnormal          917    138 [+ or -] 55
                           No interp         132
PFA-100, Col/epinephrine   Normal          10,514   119 [+ or -] 25
                           Abnormal          858    211 [+ or -] 63
                           No interp         165

                                             N      % Aggregation,
                                                    Mean [+ or -] SD

Platelet Works, ADP        Normal            605     96 [+ or -] 3
                           Abnormal           31     43 [+ or -] 32
                           No interp          11
Platelet Works,            Normal            455     90 [+ or -] 10
  collagen                 Abnormal          111     45 [+ or -] 23
                           No interp          10

                                             N       % Inhibition,
                                                    Mean [+ or -] SD

PlateletMapping, ADP       Not inhibited     366     14 [+ or -] 14
                           Inhibited         195     52 [+ or -] 23
                           No interp         791
PlateletMapping,           Not inhibited     469     11 [+ or -] 11
  arachidonic acid         Inhibited         104     47 [+ or -] 26
                           No interp         735

                                           Tirofiban

Assay                      Interpretation    N      % Aggregation,
                                                   Mean [+ or -] SD

Optical aggregation,       Normal             47    71 [+ or -] 23
  epinephrine              Abnormal         1780     7 [+ or -] 8
                           No interp         104
Optical aggregation, ADP   Normal             60    73 [+ or -] 21
                           Abnormal         1842    13 [+ or -] 13
                           No interp          79

                                             N          Ohms,
                                                   Mean [+ or -] SD

Impedance aggregation,     Normal              5   12.2 [+ or -] 5.3
  ADP                      Abnormal          475   0.6 [+ or -] 1.3
                           No interp          25

                                             N       Closure sec,
                                                   Mean [+ or -] SD

PFA-100, Col/ADP           Normal            118    100 [+ or -] 43
                           Abnormal        10,979   298 [+ or -] 16
                           No interp         153
PFA-100, Col/epinephrine   Normal            125    139 [+ or -] 49
                           Abnormal        11,015   299 [+ or -] 10
                           No interp         214

                                             N      % Aggregation,
                                                   Mean [+ or -] SD

Platelet Works, ADP        Normal             38    86 [+ or -] 13
                           Abnormal          602    35 [+ or -] 24
                           No interp          11
Platelet Works,            Normal             89    84 [+ or -] 12
  collagen                 Abnormal          481    28 [+ or -] 17
                           No interp          10

                                             N      % Inhibition,
                                                   Mean [+ or -] SD

PlateletMapping, ADP       Not inhibited       6    80 [+ or -] 39
                           Inhibited         561     95 [+ or -] 8
                           No interp         778
PlateletMapping,           Not inhibited      71    29 [+ or -] 15
  arachidonic acid         Inhibited         495    61 [+ or -] 19
                           No interp         730

Abbreviations: ADP, adenosine diphosphate; Col-ADP,
collagen-adenosine diphosphate; Col-epinephrine, collagen-epinephrine;
interp, interpretation;PFA-100, Platelet Function Analyzer 100;SD,
standard deviation.
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Author:Chandler, Wayne L.; Brown, Alan F.; Chen, Dong; Moser, Karen; Olson, John D.; Pham, Huy Phu; Smock,
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Report
Date:Apr 1, 2019
Words:5200
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