Annals of Clinical & Laboratory Science 38:247-253 (2008)
© 2008 Association of Clinical Scientists
Variables that Affect Platelet Function Analyzer-100 (PFA-100) Closure Times and Establishment of Reference Intervals in Korean Adults
Young-Uk Cho,
Seongsoo Jang,
Chan-Jeoung Park and
Hyun-Sook Chi
Department of Laboratory Medicine, University of Ulsan, College of Medicine, and Asan Medical Center, Seoul, Korea
Address correspondence to Hyun-Sook Chi, M.D., Ph.D., Department of Laboratory Medicine, Asan Medical Center, 388-1 Poongnap-dong, Songpa-gu, Seoul, Korea; tel 82 02 3 010 4502; fax 82 02 478 0884; e-mail hschi{at}amc.seoul.kr.
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Abstract
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The PFA-100 is an in vitro system for measurement of platelet function in whole blood. To establish reference intervals in the Korean population, we investigated the relationships between PFA-100 closure times and gender, ABO blood group, citrate concentration, and time of blood collection in 120 well-characterized healthy Korean adults. Gender did not affect closure time values. Blood group O was associated with longer collagen/epinephrine (CEPI) and collagen/ADP (CADP) closure times than non-O groups (p <0.0001 for both). Closure times were shorter in samples obtained in the morning vs the afternoon. Regression analysis showed an association between CEPI and CADP closure times (p <0.0001), but there were no associations between PFA-100 results and WBC count, hemoglobin, hematocrit, platelet count, and platelet-associated parameters. The reference intervals for CEPI and CADP closure times of 3.2% citrated blood were 80–162 and 64–121 sec, respectively. The reference intervals for PFA-100 closure times in healthy Korean adults were similar to those in Western populations. Flow obstruction was observed in 31 of 480 (6.5%) measurements, but routine performance of duplicate measurements was otherwise unnecessary because of acceptable CV values. PFA-100 closure time should be interpreted with consideration of the patient’s clinical presentation and factors such as ABO blood group and diurnal variation.
Keywords: PFA-100, healthy Korean adults, reference intervals, platelet function test
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Introduction
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Platelets play a pivotal role in both hemostasis and thrombosis. Primary hemostasis involves platelet adhesion to subendothelial tissue of the injured vessel wall. Under high shear stress, von Willebrand factor (vWF) acts as the mediator for both platelet adhesion and aggregation. Adhesion of platelets to collagen-containing lesions in the vessel wall results in activation of the platelets, release of various platelet activating mediators, and ultimately to augment platelet aggregation and form a primary hemostatic plug.
Accurate measurements of platelet function are important, both for screening platelet dysfunction and for monitoring response to anti-platelet therapy. Since its introduction, platelet aggregometry has become the standard method for testing platelet function. Conventional platelet aggregometry has many limitations including being time- and labor- intensive and operator-dependent, and requiring the preparation of platelet-rich plasma. Furthermore, the testing and interpretation require considerable experience [1].
Recently, a new platelet function analyzer (PFA-100, Dade Behring) was introduced that simulates in vivo haemostatic plug formation. This simple, rapid, in vitro method aids in the detection of platelet dysfunction [2]. Briefly, citrated whole blood is aspirated at a high shear rate from a sample reservoir through a 150 µm aperture in a membrane coated with collagen and epinephrine (CEPI) or collagen and ADP (CADP). Mediated by vWF, platelets adhere to the collagen on the membrane, which then activates the platelets, causing them to aggregate in and around the aperture, a process that eventually occludes the aperture. Results of PFA-100 testing are reported as closure times, or the time in sec required for the aperture to occlude. Closure time provides a measure of overall platelet-associated primary hemostasis, with shorter closure time indicative of higher platelet function.
Several pre-analytical variables affect PFA-100 test results [3–12]. Closure times depend on the plasma vWF level, citrate concentration, ABO blood group, and time of blood collection. In addition, a low platelet count or low hematocrit may lead to prolongation of closure time [7–10]. Most studies that use the PFA-100 system have been performed in Western countries. Therefore, the reported reference intervals for PFA-100 closure times are valid primarily for individuals with ethnic backgrounds found in Western countries. Venous thromboembolism is less frequent among Asians than Caucasians, for incompletely understood reasons [13–15]. Hence, there may be ethnic differences that influence the pathophysiology of hemostasis and thrombosis.
As clinical laboratory tests require cut-off levels derived from reference intervals, it is essential to establish accurate reference intervals prior to diagnostic use of new techniques. To determine PFA-100 reference intervals in the Korean population, we evaluated CEPI and CADP closure times in 120 well-characterized healthy Asian (Korean) adults. We also studied the relationships between PFA-100 closure times and gender, ABO blood group, citrate concentration, and time of blood collection in these subjects.
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Materials and Methods
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Selection of healthy subjects.
One hundred twenty apparently healthy Korean adults (>18 yr old) were enrolled in this study. The number of 120 is the recommended minimum for general practice in CLSI (formerly NCCLS) guidelines [16]. All subjects were medical students or members of the hospital staff. Equal numbers of males and females were included. About 30% of the subjects had blood group O, which is close to the proportion among the general population in Korea. Normal platelet function of each specimen was supported by results of a standardized bleeding risk assessment (SBA) questionnaire [17]. The ABO blood group of each subject was also obtained from the SBA questionnaire. Blood specimens were collected from each subject, and a complete blood count (CBC) was determined using a hematology analyzer (Sysmex XE 2100, Sysmex Corp., Kobe, Japan).
All subjects were ostensibly healthy individuals with no previous history or laboratory results indicative of platelet dysfunction. Individuals were excluded if they gave a history of bleeding disorders, liver disease, or renal insufficiency, or had taken aspirin, aspirin-containing compounds, ibuprofen, antibiotics, antihistamines, non-steroidal anti-inflammatory drugs, oral anticoagulants, or any other drugs that affect platelet function. Other exclusion criteria included use of DDAVP (1-deamino-8-D-arginine vasopressin), blood products, or antifibrinolytics within 8 days prior to sample collection, current pregnancy, platelet count <150,000 /µl, or hematocrit <35%. Each subject provided written informed consent, and the study protocol was approved by the Institutional Review Board of Asan Medical Center.
Specimen collection.
Blood samples were drawn from each subject’s antecubital vein using the BD Vacutainer system (Becton-Dickinson, Plymouth, UK). The initial small amount of whole blood was collected into EDTA collection tubes for CBC determination. Samples for PFA-100 testing were drawn into 0.109 M (3.2% w/v) and 0.129 M (3.8% w/v) sodium citrate collection tubes and stored at room temperature prior to testing. Hemolyzed or clotted samples were not used. Half of the samples were obtained in the morning (between 8:30 and 11:00 AM) and the other half in the afternoon (between 2:00 and 5:30 PM).
PFA-100 testing.
Within 4 hr of blood collection, closure times were determined on the PFA-100 system using citrated whole blood with single lots of CEPI and CADP cartridges, according to the manufacturer’s instructions. Measurements were performed in duplicate and the mean value was calculated. Testing was repeated if one replicate was non-measurable and the other was <150 sec with the CEPI cartridge or <100 sec with the CADP cartridge, or if one replicate was >2 times the value of the other replicate. Also, in case of flow obstruction, measurements were repeated from the same sample.
Statistical analysis.
The D’Agostino-Pearson test for normality was used to analyze the values. As not all values showed a normal distribution, data were presented as either median and range or as mean ± SD. Paired and unpaired data were compared using the Wilcoxon signed-rank test and the Wilcoxon rank-sum test, respectively. Reference intervals were derived from nonparametric 95% intervals [16]. Spearman rank correlation coefficients were calculated to evaluate association between closure time values and CBC parameters. Differences between duplicate measurements of all CEPI and CADP closure times were expressed as coefficient of variation (CV). Differences in frequencies were checked by Chi-square test. All statistical analyses were performed using MedCalc version 9.1 (MedCalc Software, Mariakerke, Belgium). A p value of <0.05 was considered statistically significant.
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Results
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The 120 subjects comprised 60 males and 60 females, with 50% of each providing morning and afternoon samples. The median age of the female subjects (29 yr) was less than the male subjects (32 yr), but the difference was not statistically significant (p = 0.061). Since the frequency of blood group O is 28% in Koreans, we included 36 subjects (30%) with blood group O. Of each set of samples assorted by gender and time of collection, 30% were from individuals with blood group O. For example, of blood collected in the morning from 30 males and 30 females, 9 of each had blood group O.
An overall summary of results obtained for median closure times is listed in Table 1
. Male and female blood samples collected in 3.2% citrate tubes did not have significantly different closure times in either of the cartridges. Compared with non-O blood groups, blood group O was significantly associated with prolonged CEPI and CADP closure times (p <0.0001 for both). CEPI and CADP closure times in blood collected into 3.2% citrate tubes during the afternoon were longer than those of blood collected in the morning, but the difference was not significant. Linear regression analysis revealed a significant association between CEPI and CADP closure times in 3.2% citrate tubes (r = 0.819, p <0.0001). Closure times did not correlate significantly with leukocyte count, hemoglobin, hematocrit, or platelet count. Platelet-associated parameters such as PDW, MPV, P-LCR, and PCT, also did not significantly affect closure times (data not shown).
Blood collected into 3.8% buffered sodium citrate had significantly longer closure times than blood collected into 3.2% citrate (p <0.0001 for both CEPI and CADP). Closure times of blood collected into 3.8% citrate tubes showed the same findings as those in 3.2% citrated blood. The only exception was that CEPI closure times in blood collected into 3.8% citrate tubes in the afternoon were significantly longer than those collected in the morning (p = 0.032) (data not shown).
For samples obtained in 3.2% citrated blood, the reference intervals (95% central interval) of CEPI and CADP closure times determined in the morning were 79–148 and 63–110 sec, respectively, whereas the intervals for specimens collected in the afternoon were 85–170 and 66–127 sec, respectively. The reference intervals of 3.2% citrated blood, regardless of collection time (n = 120), were 80–162 sec for CEPI and 64–121 sec for CADP cartridges.
Duplicate testing of PFA-100 with CEPI cartridges exhibited relatively consistent CV values according to test conditions (Table 2). With 3.2% citrated blood, the CVs (mean ± SD) were 5.5±4.2% for CEPI and 4.3±3.9% for CADP cartridges. We found that 5.0% (n = 6) of 120 duplicates with CEPI cartridges exceeded 15% CV, as did 1.7% (n = 2) of 120 duplicates with CADP cartridges.
Because only prolonged closure times have clinical relevance, including defects in primary hemostasis and prediction of bleeding tendency, we arbitrarily defined "upper aberrations" as one result of duplicate measurements being within reference limits and the other exceeding the upper limit of the corresponding reference interval. We found that the frequency of "upper aberrations" was 8.3% for CEPI cartridges and 4.2% for CADP cartridges (Table 2
). That is, most duplicate measurements were within the reference intervals, although there was some degree of variation. A major proportion of "upper aberrations" occurred in blood group O samples (60% for CEPI cartridges and 100% for CADP cartridges) (Table 2).
The PFA-100 instrument can detect a sudden stoppage of blood flow during the test. We found that the frequency of such flow obstructions was 8.3% (20/240) in CEPI and 4.6% (11/240) in CADP cartridges, making the overall frequency of flow obstruction 6.5% (31/480). Flow obstruction was not affected by the type of PFA-100 cartridges, citrate concentration, time of blood collection, or test sequence (data not shown). In cases of flow obstruction, we repeated the tests using the same blood samples, and obtained measurable PFA-100 values in all instances.
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Discussion
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Clinical performance of the PFA-100 device to screen for platelet dysfunction may be influenced by pre-analytical variables. Previous studies have found that age and gender have no effects on PFA-100 closure times [8,18], although men older than 55 yr appeared to have shorter closure times than younger men [18]. Diurnal variations in closure time values have been reported, with samples collected in the morning having shorter closure times than those collected the afternoon, particularly with the CEPI cartridge [4]. Closure times are longer for blood group O samples than for other blood groups [4–8], and are longer for blood samples collected in 3.8% vs 3.2% sodium citrate [3,7–9]. Although PFA-100 closure times are prolonged when platelet count or hematocrit is significantly decreased [7–10], the cut-off value for abnormality of either has not been determined. PFA-100 closure times are usually prolonged when platelet count is <50,000/µl or hematocrit is <25% [9]. In this context, the manufacturer notes that closure times above the laboratory’s established cut-off may reflect reduced platelet function caused by reduced hematocrit (<35%) or platelet count (<150,000/µl).
We confirmed previous findings that PFA-100 closure times in healthy adults are not influenced by gender and do not correlate with leukocyte count, platelet count, hematocrit, or platelet-associated parameters of routine CBCs [19]. As expected, individuals with blood group O had longer PFA-100 closure times than those with other blood groups, likely because vWF levels are lower in blood group O than in other blood groups [20,21]. PFA-100 closure times are strongly dependent on the plasma vWF level [7–9,12]. During primary hemostasis, vWF serves as an adhesive molecule that tethers the platelet to exposed collagen at sites of vascular injury. Thus, lower levels of vWF affect the formation of a hemostatic plug and typically prolong PFA-100 closure time.
We confirmed that PFA-100 closure time is prolonged in blood samples obtained in the afternoon compared to those obtained in the morning. The results suggest that platelet function in general might show diurnal variation, being higher in the early morning hours and lower in the afternoon. As expected, we found that blood anticoagulated with 3.8% buffered sodium citrate yielded longer closure times than blood anticoagulated with 3.2% citrate. The prolongation of PFA-100 closure time in 3.8% citrate is likely related to its stronger chelation of calcium [3], consistent with the dependence of hemostatic reactions on calcium concentration.
Despite many evaluations of the clinical performance of the PFA-100 device in Western populations, few studies have determined reference intervals under standardized conditions and in a large number of healthy adults (Table 3). Using 0.105 M (3.2%) sodium citrate blood collection tubes, the reference CEPI and CADP closure times in 309 German adults were found to be 82–150 and 62–100 sec, respectively [19]. In 3.8% citrate tubes, the reference intervals were longer, 94–191 sec for CEPI and 72–120 sec for CADP cartridges [2]. It is unclear, however, if the samples in either study were collected in the morning or afternoon. In a recent study using blood samples from 120 German adults, the reference intervals for closure times in the morning were 93–223 sec for CEPI and 65–117 sec for CADP cartridges [22]. These wide ranges of closure times are likely due to collection of the samples into 3.8% citrate tubes.
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Table 3. Reference intervals of PFA-100 closure times from 3 reports that studied large numbers of healthy individuals.
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Most hematology laboratories in Korea use 3.2% sodium citrate tubes, and more laboratories throughout the world are switching to 3.2% citrate tubes. Therefore it is more practical to establish reference intervals for PFA-100 closure times in 3.2% rather than 3.8% citrated blood. We found that the reference intervals of 3.2% citrated blood, irrespective of collection time and ABO type, were 80–162 sec for CEPI and 64–121 sec for CADP cartridges, findings comparable to those obtained previously using 3.2% citrate tubes [19], although the upper limits of closure time were higher in our study. Taken together, our findings show that ethnic differences in platelet function status, as evidenced by PFA-100 closure times, are minimal between Western and Oriental populations. In using the PFA-100 system as a routine clinical test in Korea, attention should be paid to the sampling conditions. For example, a patient with borderline platelet dysfunction scheduled for surgery in the afternoon might go unnoticed if tested in the early morning.
It is unclear if and when duplicate measurements in PFA-100 testing are required. An early study in healthy controls found that only 1.5% of samples needed retesting due an unacceptable variation of more than 20% between the duplicates (0.8%) or to test interruption resulting from flow obstruction in one of the two replicates (0.7%), suggesting that there was no need for routine duplicate testing [19]. More recently, in patients with a history of bleeding, the mean CV values were 7.1% for CEPI and 5.7% for CADP cartridges, with 16% (CEPI) and 9% (CADP) of duplicates showing a 
15% difference from their means [23]. Another recent study reported 15% differences from the mean in 11.7% of samples using CEPI and 10.6% using CADP cartridges [22]. These findings strongly suggest a need for duplicate testing to obtain reliable values. In the original study in characterizing the diagnostic performance of the PFA-100 system, no clear benefit of duplicate testing on platelet function classification could be shown [2]. In our study, the mean CV values using 3.2% citrated blood were 5.5% for CEPI and 4.3% for CADP closure times. The frequency of CVs >15% was 5.0% with CEPI and 1.7% with CADP cartridges, somewhat lower than the 4.7–12.8% with CEPI and 3.7–10.3% with CADP cartridges observed previously [2,3,9,23].
Most duplicates in our study were within the reference ranges. The frequency of "upper aberrations" was minimal, with the majority observed in blood group O samples. These findings indicate that the distribution of closure times found in blood group O individuals is shifted slightly to higher closure times when compared to the non-O groups. We assured that in the normal population used for the generation of these ranges sufficient type O was represented, so the effect of the ABO-type is embedded in the overall reference ranges, avoiding a need for ABO type-specific ranges.
The phenomenon called flow obstruction may be caused by microthrombi in the sample or particulates introduced into the sample from the environment. The use of 0.106 M unbuffered sodium citrate has been reported to generate a significantly higher frequency of flow obstruction (7.0% in CEPI and 11.2% in CADP cartridges) than buffered sodium citrate [3]. We observed flow obstruction in 6.5% of 480 measurements, a frequency lower than that of unbuffered citrate (overall 9.1%), but higher than with various concentrations (1.5–1.9%) of buffered citrate [3]. In our study, blood was collected into buffered citrate, followed by mixing of blood and anticoagulant to avoid clot formation, with all tests performed within 4 hr after blood collection. Thus, the reason for the relatively high occurrence of flow obstruction in this study is unclear. However, all samples showing flow obstruction produced reasonable values after repeated testing without resampling. Our results suggest that routine duplicate testing of all samples is unnecessary. Only individuals having unexpected PFA-100 results and samples generating flow obstructions should be retested, which reduces the costs incurred by the testing.
This study has several limitations. Although we attempted to obtain a representative group of healthy Korean adults, we could not avoid bias because our sample consisted predominantly of young subjects. Younger men have been reported to have longer closure times than older men [18]. However, since age has not been found to influence PFA-100 results significantly, a shortage of relatively older subjects likely does not limit the relevance of our data. Although we meticulously selected the study subjects, based on a questionnaire validated in >10,000 elective surgery patients, we cannot exclude that a few subjects with mild platelet dysfunction due to an effect of anti-platelet drug intake might have been included. In addition, we did not compare PFA-100 test results with platelet aggregometry, the standard method of platelet function testing. Furthermore, since we did not measure plasma vWF levels, which affect PFA-100 closure times, we could not verify the cause of prolonged closure times that exceeded the normal ranges.
In conclusion, this report is the first to assess the performance of the PFA-100 instrument in a well-characterized healthy Asian population. The external variables that influence PFA-100 closure times in Western populations also influence closure times in Korean subjects. The reference intervals obtained in our study are comparable to those in Western populations. Our findings suggest that all blood samples do not need to be tested in duplicate, except for samples in which flow obstruction occurs or in case of unexpected results. By standardizing pre-analytical test conditions, the PFA-100 can become a first-line tool for screening of platelet dysfunction. Our findings should be the basis for clinical use of the PFA-100 technology in Korea.
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Acknowledgements
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This study was supported by a grant from Dade Behring Marburg GmbH–A Siemens Company, Marburg, Germany. We acknowledge the technical assistance of Min-Sook Cho and Min-Young Han.
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Abstract
Introduction
