International Journal of Laboratory Hematology The Official journal of the International Society for Laboratory Hematology
ORIGINAL ARTICLE
INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY
Assessment of spontaneous platelet aggregation using laser light scattering in healthy subjects: an attempt to standardize S. SUZUKI* ,† , H. KUDO † , T. KOYAMA*
*Laboratory Molecular Genetics of Hematology, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan † Department of Clinical Laboratory Medicine, Faculty of Health Science Technology, Bunkyo Gakuin University, Bunkyo-ku, Tokyo, Japan Correspondence: Takatoshi Koyama, MD, PhD, Graduate School of Health Care Sciences, Tokyo Medical and Dental University 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. Tel./Fax: +81-3-5803-5882, E-mail: [email protected] doi:10.1111/ijlh.12208
Received 20 November 2013; accepted for publication 10 February 2014 Keywords Platelet, aggregation, spontaneous platelet aggregation (SPA), P-selectin, activated glycoprotein IIb/IIIa
S U M M A RY Introduction: When measuring platelet aggregation using laser light scattering, small aggregates forming without the addition of agonists may be observed. This event is called ‘spontaneous platelet aggregation (SPA)’. The platelet hyperactivity observed in arterial thrombotic diseases can be detected with relative ease by measuring SPA. Standardization is urgently needed because of differences between measurement conditions among various laboratories. Methods: We conducted a systematic study of factors that affect SPA measurement, compared SPA results to flow cytometry detection of surface antigens expressed on activated platelet membranes (P-selectin, activated glycoprotein IIb/IIIa), and determined conditions that yield stable measurements. Results and Conclusions: We evaluated results from 125 healthy volunteers and established conditions for a stable measurement of SPA. As the occurrence of SPA tended to increase with age, we determined conditions valid for subjects aged 20–60 years. Blood should be collected using a syringe, and the sample should be prepared after allowing the whole blood to rest for 30 min after collection. To isolate platelet-rich plasma, a 2-mL tube should be used and centrifuged at 150 g. The sample should be stored at room temperature, the platelet count of the sample should be (250 10) 9 109/L, and the measurement should be completed within 90 min of blood collection.
INTRODUCTION Platelets play an important role in hemostasis, and platelet hyperactivity is a factor in thrombus formation. Platelet hyperactivity is observed in arterial thrombotic diseases such as myocardial infarction and 676
cerebral infarction [1–4]. Therefore, understanding platelet hyperactivity is useful for diagnosis and treatment. If platelets are hyperactive, various changes occur in association with increased aggregatory reactivity and the release of contents of platelet granules in vivo. © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 676–685
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Therefore, historically, these items have been used as indicators to evaluate platelet hyperactivity. However, this method requires very expensive equipment, and the procedure itself is expensive, making it an uncommonly used method. One method of testing platelet function is the optical density (OD) method devised by Born [5]. This method was once the most commonly used method of undertaking a platelet-aggregation test; it is effective for diagnosing platelet dysfunction but has low sensitivity for detecting small aggregates of platelets and thus is difficult to use for evaluating platelet hyperactivity [6, 7]. However, particle-counting platelet aggregometers [8], which detect platelet aggregates using forward scattering of light with high-detection sensitivity and can even detect aggregates formed by only a few platelets, have made it possible to measure spontaneous platelet aggregation (SPA) that the OD method cannot detect. SPA is defined as small aggregates that form in vitro through stirring alone without the addition of agonists. Studies have reported that the number of these aggregates increases if platelets are hyperactive [9–12]. The laser light scattering (LS) method is based on the principle that the intensity of particle scattering is proportional to the square of the particle diameter and that the detected intensity of scattering can be used to obtain information regarding the number and size of aggregates in platelet-rich plasma (PRP). In this study, we used the LS method with a platelet aggregometer to explore simpler methods of detecting platelet hyperactivity by assessing platelet activity via detection of small platelet aggregates. However, it is known that measured values of SPA change depending on differences in conditions between blood collection and measurement. To enable wide application of measured values of SPA in clinical laboratories, they must have good precision and accuracy. This warrants standardization of conditions of blood collection and measurement [13–15]. Therefore, we systematically evaluated factors that influence SPA between blood collection and measurement to determine conditions that yield stable measurements. For confirmation, we compared SPA detection with flow cytometry (FCM) detection of activated glycoprotein (GP) IIb/IIIa (a fibrinogen receptor) and P-selectin (which reflects the release of © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 676–685
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a-granules). In addition, we attempted to determine conditions for measuring SPA that would be valid for 2 age groups of subjects in their 20 s and those aged ≥ 30 years.
M AT E R I A L S A N D M E T H O D S Subjects This study was approved by the ethics committee of Bunkyo Gakuin University (Tokyo, Japan). It was conducted on venous blood collected from 125 healthy, consenting students and faculty members (44 men and 81 women; 21–60 years). Blood from individuals taking drugs that could affect platelet aggregation was not used. Blood sampling Polypropyrene syringes and evacuated polyester tubes of 13.2φ 9 78 mm containing citrate were purchased from Terumo, Tokyo, Japan. Fasting blood samples were collected between 10:00 and 11:00 am to avoid diurnal variations in platelet aggregation [16]. Moreover, platelets can be activated by blood collection, and there can be individual differences in collection methods. Therefore, two experienced technicians collected all blood samples. Blood with air bubbles and blood that was difficult to collect was not used; only blood that was readily collected within 30 s of puncture was used. 12 mL of blood was collected from the median antebrachial vein using a tourniquet and a syringe with a 21-G needle. Collection was completed within 30 s of puncture. When SPA was measured, the initial 2 mL of blood collected was discarded, and the remaining blood was used. After the initial 2 mL of blood was discarded, the rest was divided into 2- and 5-mL tube without or with a top seal in a balanced order (i.e., into an evacuated tube first in some subjects and second in other subjects). Procedure and methods of SPA measurement
Preparation of measurement samples One unit of 109 mM sodium citrate had been added per nine units of blood. The tube was gently inverted
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three times and the blood left to rest at 25 °C for 30 min. PRP was isolated by centrifugation of blood at 150 g for 10 min at 25 °C. The remaining blood was centrifuged at 2000 g for an additional 10 min at 25 °C to isolate platelet-poor plasma (PPP). PPP was used to dilute PRP from the same subject to prepare a sample with a platelet count of (250 10) 9 109/L. A multi-item automatic blood cell analyzer (XS-1000i; Sysmex, Tokyo, Japan) was used to calculate the platelet count. Polypropyrene tubes (12φ 9 75 mm) for flow cytometric analysis (Beckman Coulter, Pasadena, CA, USA) were used during the preparation and handling of PRP.
SPA measurement method Spontaneous platelet aggregation was measured using a laser-scattering particle-counting platelet aggregometer (PA-20 of 2 channels and PA-200 of 4 channels; Kowa, Tokyo, Japan). To compare data, we ensured that there was no difference between channels of the device. The sample for measurement was dispensed into a cuvette with a stirring bar. After preheating the sample at 37 °C for 2 min, aggregation was measured for 10 min while only stirring at 2.43 g and without adding an agonist. SPA was evaluated using the maximum value of total light scattering intensity by small aggregates for a 10 min (SMax); SMax ≥ 2.0E4 mV 9 count was considered SPA positive (Figure 1). Other platelet-detection methods in comparison with SPA Rates of expression of activated GPIIb/IIIa and P-selectin, which are expressed on the surface of activated platelet membranes, were measured by FCM using monoclonal PAC-1 antibodies and anti-CD62P antibodies [17]. The flow cytometer used was an Epics XL (Beckman Coulter). A testing center (SRL, Tokyo, Japan) was commissioned to measure levels of bthromboglobulin and platelet factor 4, which are markers of platelet activation and indicators of the release reaction [18], using Asserachrom PF-4 and Asserachrom b-TG (Roche Diagnostics, Tokyo, Japan), respectively. Adenosine diphosphate (ADP) aggregation at a final concentration of 2 lM and collagen aggregation at a final concentration of 1 lg/mL were
Figure 1. SPA-positive record of time-dependent formation of platelet aggregates measured by laser light scattering (LS) method and optical density (OD) method in the absence of aggregation inducers as compared with ADP-induced traces. The total LS intensity reflected by small aggregates containing four to nine platelets increased slightly in a timedependent manner (-(S)-, blue curve), and there was virtually no change in the total LS intensity reflected by medium containing 10 to 99 platelets (-(M)-, green curve) and large containing over 100 platelets (-(L)-, red curve) aggregates. No increase of OD method (-(T)-) was observed in subjects. (1) We measured SPA as the maximum value of total LS intensity by small aggregates for a 10 min (SMax); SMax ≥ 2.0E4 was considered SPA-positive. This case showed 12E4 of SMax. (2) shows traces of 2 lM of ADP-induced platelet aggregation.
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measured using the PA-20 and PA-200 systems using the OD method.
indication for platelet aggregates and large and immature platelets.
Items assessed
Thirty-minute sample resting period before measurement
Experiments presented below were performed using the above-mentioned blood sampling method and SPA measurement procedures. Only assessed items were changed; all other conditions were the same. To avoid the influence of time, SPA measurements were undertaken simultaneously. To avoid effects of platelet activation and pH changes, all experiments were performed in a room maintained at 25 1 °C, and samples were stored in tightly sealed polypropylene test tubes.
Method of blood collection and anticoagulation Effects of the blood collection method (syringe or vacuum tube containing citrate) and the concentration of the anticoagulant sodium citrate (106 or 109 mM) on SPA were assessed using blood samples collected from the same subject by measuring and comparing SPA as well as surface antigen expression. Osmotic pressure of PRP was measured with Osmo STAD OM-6040 (Arkray, Kyoto, Japan).
The stability of allowing whole blood to rest for 30 min immediately after collection was compared with the stability of leaving a prepared measurement sample of blood from the same subject to rest for 30 min based on SPA and surface antigen expression.
Storage temperature of samples After storing the prepared measurement samples from the same subject for 30 min at 4, 25, or 30 °C, SPA was simultaneously measured to evaluate effects of storage temperature on SPA.
Platelet count Measurement samples were prepared with four platelet concentrations (100, 250, 300, 400) 9 109/L with blood from the same subject. SPA was simultaneously measured to evaluate the effects of the platelet count on SPA.
Centrifugal force Blood from the same subject was dispensed into two blood-collection tubes of the same size (2 and 5 mL) that contained 0.2 and 0.5 mL of the anticoagulant. Tubes were simultaneously centrifuged at 150 g for 10 min at 25 °C. Measurement samples were prepared, and SPA was simultaneously measured. Effects of centrifugal force while separating PRP were examined using SPA along with the platelet large-cell ratio (P-LCR), mean platelet volume (MPV), and platelet distribution width (PDW) of the measurement sample. The relative centrifugal force (RCF) at this time was calculated as the average of the centrifugation force (g) applied to the bottom of the test tube and that applied to the liquid surface. The P-LCR, MPV, and PDW were measured using the XS-1000i multi-item automatic blood cell analyzer. The P-LCR indicates the percentage of large platelets with a volume > 12 fL. An increase in the parameter may be an
© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 676–685
Time-dependent changes Measurement samples prepared from the same subject were measured 60, 90, 120, and 180 min after collection to examine the effect of the time elapsed between collection and measurement on SPA.
Age-related differences in SPA Using the same method of blood collection, SPA values from samples that had been prepared and measured were examined according to age group. Statistical analyses SPSS ver18 (IBM, Tokyo, Japan) was used for calculations. Correlation analyses were performed using Spearman’s correlation. Tests of significance were conducted using repeated-measures ANOVA and Wilcoxon
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t-tests. P < 0.05 was considered significant. The data were shown with mean SEM.
R E S U LT S The correlation between SPA and other methods of detection of platelet activation is presented in Table 1. Correlations with SPA were determined for CD62P and PAC-1; correlation coefficients were 0.440 and 0.705, respectively. SPA values for the syringe method of blood collection were significantly lower than those of the vacuum-tube method, and the expression of P-selectin and activated GPIIb/IIIa was lower, although not significantly so (Table 2-1). No significant difference in SPA values was observed between 106 and 109 mM concentrations of the anticoagulant sodium citrate, which are both commonly used. pH levels of these samples at the time of measurement were 7.91 and 7.89, respectively, and there was no significant difference in osmotic pressure (Table 2-2). In the experiment focusing on effects of centrifugal force on SPA, RCF applied to 2- and 5-mL test tubes was 139 and 124 g, respectively, and SPA was significantly higher for the 2-mL tube, which experienced stronger RCF. In contrast, the P-LCR, MPV, and PDW were significantly higher for the 5-mL tube, which experienced weaker RCF (Table 2-3). In the experiment with the 30-min resting period between blood collection and measurement, allowing the blood to rest as whole blood resulted in a significantly lower SPA than preparing the sample by isolating PRP before the resting period (Table 2-4). Moreover, the expression of activated GPIIb/IIIa was
Table 1. Correlation between SPA and other methods of detection of platelet activation
Platelet aggregation Platelet activation markers
Class
n
r
P value
ADP (%) Collagen (%) b-TG (ng/mL) PF-4 (ng/mL) CD62P expression (%) PAC-1 expression (%)
68 68 35 35 45
0.277 0.172 0.199 0.370 0.440
0.022 0.162 0.251 0.029 0.002
45 0.705 0.000
Table 2. Effects of (1) blood collection method on platelet activation (2) anticoagulant sodium citrate on SPA (3) Centrifugal force on platelet activation (4) Platelet activation that 30-min resting period gives (1)* Vacuum tube
P value
Collection method
Syringe
SPA (SMax E4) CD62P expression (%) PAC-1 expression (%)
2.95 0.75 3.56 0.82 1.26 0.17 1.40 0.21
0.028 0.611
1.35 0.29 1.71 0.28
0.120
(2)† Concentration pH Osmotic pressure (mOsm/kg) SPA (SMax E4)
106 mM (3.13%)
109 mM (3.20%)
P value
7.91 0.05 7.89 0.06 0.206 283.3 1.69 283.3 1.51 0.922 4.41 1.16
4.59 1.05 0.167
(3)‡ Blood-collection tube RCF (9g)
2 mL
5 mL
139
129
SPA (SMax E4) P-LCR (%) MPV (fL) PDW (fL)
1.88 14.39 8.83 9.13
0.41 1.43 0.27 1.16 20.79 1.51 0.15 9.89 0.34 0.29 10.39 0.34
P value 0.034 0.000 0.002 0.000
(4)§ Prepared sample SPA (SMax E4) CD62P expression (%) PAC-1 expression (%)
Whole blood
P value
4.97 0.75
4.04 0.60 0.037
6.56 0.67
5.05 1.01 0.099
33.35 2.42
29.41 1.87 0.028
PLC-R, platelet large-cell ratio; MPV, mean platelet volume; PDW; platelet distribution width. *n = 6. †n = 8. ‡n = 16. §n = 12.
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significantly lower, and the expression of P-selectin was lower, but not significantly so. Platelets with a low SPA value were not affected by the storage temperature, but platelets that were
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SPA positive and moderately active revealed higher SPA under lower storage temperatures (Figure 2-1). In the experiment focusing on the effect of platelet count on SPA, higher platelet counts tended to be
Figure 2. Effects of measurement condition on SPA. Experiments were performed using the same method of blood sampling and SPA measurement procedures. Only assessed items were changed; all other conditions were the same. SPA values from samples were examined according to 2 age group (subjects in their 20s and subjects aged ≥ 30 years). The dark gray column expresses SPA values from subjects in their 20s, and the bright gray column expresses subjects aged ≥ 30 years, and the line graph expresses overall mean. Subjects aged ≥ 30 years were comprised of 1-2 subjects chosen from every age group. (2-1): Effect of storage temperature on SPA. After storing prepared measurement samples from the same subject for 30 min at 4, 25, or 30 °C. Subjects in their 20s were 7 peoples and subjects aged ≥ 30 years were 7 peoples. No significant difference was observed between SPA at the platelet storage temperature at 25 °C, and platelet storage temperature at 30 °C, with respect to overall mean SPA and SPA of subjects in their 20s. But a significant difference was observed in subjects aged ≥ 30 years. This suggests that sample storage at room temperature (25 °C) is suitable for both age groups. (2-2): Effect of platelet count on SPA. Measurement samples were prepared with four platelet concentrations (100, 250, 300, 400) 9 109/L with blood from the same subject. Subjects in their 20s were 6 peoples and subjects aged ≥ 30 years were 6 peoples. SPA significantly changed with the platelet count. No significant difference was observed between SPA at a platelet concentration of 250 9 109/L (which is most reactive and has the highest SPA), and platelet concentrations of 250–300 9 109/L with respect to overall mean SPA and SPA of subjects aged ≥ 30 years, but a significant difference was observed in subjects in their 20s. This suggests that a concentration of 250 9 109/L is suitable for both age groups. (2-3): Effect of time-dependent changes on SPA. Measurement samples prepared from the same subject were measured 60, 90, 120, and 180 min after collection. Subjects in their 20s were 5 peoples and subjects aged ≥30 years were 5 peoples. SPA increased significantly with longer time intervals regardless of age. SMax of ≥2.0E4 was considered to indicate SPA. Blood samples revealed only a normal SPA range for healthy individuals (2.0E4) ≤90 min after collection. This result suggests that SPA measurements should be completed ≤90 min of blood collection.
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associated with higher SPA regardless of age of the subject, and a significant difference was observed for all intervals except 250–300 9 109/L (Figure 2-2). In addition, SPA increased significantly with longer time intervals in the experiment investigating time-dependent changes until measurement of the sample (Figure 2-3). All subjects were healthy, but some samples were SPA positive (scattering intensity of 2.0E4) when measured after >90 min had elapsed since sampling. Spontaneous platelet aggregation values of 50 healthy subjects measured are presented according to age in Figure 3. The SPA-positive rate was calculated for 2 age groups of subjects in their 20s and those aged ≥ 30 years. The positive rate tended to increase with age.
DISCUSSION Activated GPIIb/IIIa and P-selectin were used as points of comparison for SPA in the present study because they significantly correlated with SPA, and are thus useful for detecting platelet activation (hyperactivity).
Figure 3. Age-related differences in SPA. SPA values from samples of 50 healthy subjects were examined according to age group. Subjects in their 20s were 10 peoples and those aged ≥ 30 years were 40 peoples. SPA-positive (SMax ≥ 2.0E4) rate was calculated for each age group, which tended to increase with age. SPA-positive rate of subjects in their 20s were 20% (2/10) and those aged ≥ 30 years were 50% (20/40). Even if the positive rate of subjects in their 20s calculated in 45 subjects, it was 20% (9/45).
It is recommended that a 19-G needle be used without a tourniquet when collecting blood for platelet-aggregation tests. The normal practice is to use a tourniquet for a minimal amount of time in order to insert the needle and then to release the pressure during blood collection to avoid hemoconcentration and activation. As it is often difficult to take blood from a Japanese female using a 19-G needle, we collected blood using a tourniquet and a 21-G needle, the most commonly used needle. We compared the syringe and vacuum-tube methods of blood collection and determined that collecting samples using a syringe affected platelet activation significantly less often compared with collecting samples with a vacuum tube, which suggests that using a syringe is a better method of blood collection. When FCM was used, the syringe method of blood collection revealed decreased expression of activated GPIIb/IIIa and P-selectin compared with that in the vacuum-tube method. This increase in SPA probably resulted because of moderate activation of platelets induced by the strong force of blood that is sucked in by the vacuum hitting the test tube wall. The World Health Organization (WHO) and Clinical and Laboratory Standards Institute (CLSI) recommend that anticoagulant sodium citrate concentrations of 3.2% (which is isotonic with blood) should be used in coagulation tests [19, 20]. However, the desirable concentration for platelet-aggregation tests is not clear. Therefore, we examined two commonly used sodium citrate concentrations (106 and 109 mM) but found no significant difference between them. A few studies [21, 22] have suggested that platelet aggregation is affected by pH, but minor changes observed in the present study were not strong enough to affect SPA. Centrifugation must be performed when conducting a platelet-aggregation test. We examined two commonly used volumes of blood mixed with anticoagulant in a collection tube (2 and 5 mL). On investigating the effect that RCF, which was used to obtain PRP, had on SPA, we determined that SPA was higher in tubes with 2 mL blood, which have a higher RCF applied to them. This result contradicts that of studies [23, 24] suggesting that a high RCF removes highly active large platelets and therefore yields low aggregation. However, the platelet large-cell ratio and the mean platelet volume were higher for 5-mL tubes, © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 676–685
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which had a lower RCF applied to them. The reason why these tubes revealed lower SPA may have been that the 2-mL tubes, with high RCF applied to them, may have experienced a large shearing stress. Collecting 5 mL blood not only yields a lower PRP volume and a larger platelet count in PRP, thereby making sample preparation for measurement more time-consuming, but also causes other inconveniences such as platelet activation during sample preparation. However, collecting 2 mL blood is convenient because it allows for a smaller amount of blood to be collected because of the large amount of PRP that can be obtained, and the platelet concentration in PRP is close to the concentration of 250 9 109/L that is to be prepared, which makes it easy to prepare samples for measurement and prevents platelet activation. Standardization of centrifugation conditions is important for standardization of the entire process, but the volume of blood collected and the size of blood-collection tube must also be taken into account. Our results suggest that in clinical laboratories, 2 mL blood should be collected and spun at 150 g. We used SPA and FCM to determine whether blood should be left to rest as whole blood or as a prepared sample during the 30 min between blood collection and measurement and found that leaving blood as whole blood yielded good SPA results. The reason for this may be that the ‘double load’ placed on blood by collection and producing PRP through centrifugation has a stronger effect on platelets and that subjecting blood to the load of PRP isolation after the load from blood collection has subsided is a more suitable method. The fact that the expression of activated GPIIb/IIIa using FCM was higher in samples that were immediately prepared also suggests that this double load induced further activation, which could have caused platelets to sensitively react to even slight stimuli, thereby causing SPA to increase. This result suggests that it is best to let the blood rest as whole blood immediately after collection. This result will be very helpful for busy clinical facilities that process a large number of blood samples because samples can be processed in batches, and there is no need to centrifuge individual samples immediately to prepare PRP. Studies [25, 26] have reported that platelet aggregation increases at low storage temperatures. We examined the effects of storage temperature on plate© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 676–685
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let activation during the 30-min resting period. We observed that preheating platelets of subjects in their 20s at 37 °C for 2 min just before platelet aggregation measurement negated any effect of storage temperature, but that storing platelets of subjects aged ≥ 30 years at 4 °C caused SPA to significantly increase and at 30 °C caused SPA to significantly decrease. Therefore, storing platelets at low temperatures controlled dissociation and enhanced aggregation [27, 28], but preheating platelets of subjects in their 20s at 37 °C for 2 min dissociated aggregates and thus did not affect SPA. In contrast, platelets of older subjects were in a moderately active state and thus may not have dissociated aggregates as easily. This result suggests that taking the age and pathological condition of patients into account, samples should be stored at room temperature (25 °C) during the 30min resting period when conducting a test. We used our room temperature of 25 °C, as in Japan, a hematological laboratory room is generally linked to outpatient rooms in a hospital and the temperature in the hospital is regulated to 25 °C. Resting for 30 min seems to be necessary to escape from stimulus of blood collection procedure and to be convenient for operators to prepare for the SPA measurement. Resting for 2 h may not be a problem for measurement of agonist-induced platelet aggregation, but as indicated in Figure 2-3, more than 1 h after collection, SPA is induced. As SPA should be measured concentratedly with reservation, hospital transport systems may not be suitable for SPA measurement. We examined effects of the platelet count of the measurement sample on SPA and found that SPA significantly changed with the platelet count. No significant difference was observed between SPA at a platelet concentration of 250 9 109/L, which is most reactive and has the highest SPA, and platelet concentrations of 250–300 9 109/L with respect to overall mean SPA and SPA of subjects aged ≥ 30 years, but a significant difference was observed in subjects in their 20s. This suggests that a concentration of 250 9 109/L is suitable for both age groups, and this is the easiest concentration to prepare in clinical settings (it is difficult to prepare a concentration of 300 9 109/L from a patient with a low platelet count). The probable reason why a difference was observed between subjects in their 20s and those aged ≥ 30 years may be that platelets of subjects in their 20s reacted sensitively to
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agitation by the stirrer during measurement depending on the platelet count, but that platelets that are already moderately activated react similarly and are not affected by minor changes in the platelet count if SPA is high. Studies [29–31] have shown that it is not necessary to adjust the platelet count of measurement samples when using conventional methods of measuring platelet aggregation (e.g., ADP and collagen aggregation). However, SPA reacts sensitively to platelet count because it measures aggregation by detecting aggregates that are too small to affect optical density, and several samples have a PRP platelet count outside the range of 250–300 9 109/L, which is why platelet count should be adjusted when using this method. We evaluated changes in SPA based on the time elapsed from blood collection to measurement. We observed that SPA increased significantly with longer time intervals regardless of age. SMax of ≥2.0E4 was considered to indicate SPA. Blood samples revealed only a normal SPA range for healthy individuals (2.0E4) ≤90 min after collection. This result suggests that SPA measurements should be completed ≤90 min of blood collection. When we measured SPA under conditions described above, we determined a larger prevalence of SPA positives (SMax ≥ 2.0E4) in subjects aged ≥ 30 years compared with subjects in their 20s, although all subjects were healthy. Only 20% subjects in their 20s were positive for SPA, whereas 50% subjects aged ≥ 30 years were positive. This age-related increase in SPA suggests the following: (i) age must be taken into account when setting standard values, (ii) platelets become unstable and increasingly reactive with age although they are
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AC K N OW L E D G E M E N T S We are very grateful to Dr Shinobu Sakamoto for appropriate support and Kumiko Yanagisawa for assisting with our experiments.
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in resting platelets from NIDDM patients is enhanced: correlation with spontaneous aggregation. Diabetes 1997;46:488–93. Eto K, Takeshita S, Ochiai M, Ozaki Y, Sato T, Isshiki T. Platelet aggregation in acute coronary syndromes: use of a new aggregometer with laser light scattering to assess platelet aggregability. Cardiovasc Res 1998;40:223–9. Satoh K, Yatomi Y, Osada T, Takeda S, Tsuyuguchi N, Kubota F, Ozaki Y. Clear visual detection of circulating platelet aggregates in acute myocardial infarction using a flow cytometer equipped with an imaging device. Platelets 2004;15:61–2. Hara K, Omori K, Sumioka Y, Aso Y. Spontaneous platelet aggregation evaluated by laser light scatter in patients with type 2 diabetes: effects of short-term improved glycemic control and adiponectin. Transl Res 2012;159:15–24. Toshima H, Sugihara H, Hamano H, Sato M, Yamamoto M, Yamazaki S, Yamada Y, Taki M, Izumi S, Hoshi K, Fusegawa Y, Satoh K, Ozaki Y, Kurihara S. Spontaneous platelet aggregation in normal subject assessed by a laser light scattering method: an attempt at standardization. Platelets 2008;19:293–9. Ozaki K. Detection of activated platelets and its clinical significance. J Jpn Soc Lab Hematol 2008;9:401–8. Satoh K, Schimizu M, Ohara S, Goto H, Sano M, Yamazaki M, Toshima H, Yamamoto M. Questionnaire survey on platelet aggregation tests in Japan. J Jpn Soc Lab Hematol 2008;9:167–77.
16. Shimizu M, Tanaka Y, Ando Y. Clinical significance and problems of flow cytometric analysis of platelet counts and platelet activation using monoclonal antibodies against platelet membrane glycoproteins. J Jpn Soc Lab Hematol 2003;4:99–108. 17. Michelson AD. Flow cytometry: a clinical test of platelet function. Blood 1996;87: 4925–36. 18. Kaplan KL, Owen J. Plasma levels of bthromboglobulin and platelet factor 4 as indices of platelet activation in vivo. Blood 1981;57:199–202. 19. World Health Organization (WHO). Use of anticoagulants in diagnostic laboratory investigations. WHO/DIL/LAB/99.1, Rev.2; 2002. 20. Adcock DM, Hoefner DM, Kottke-Marchant K, Marlar RA, Szamosi DI, Warunek DJ. Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis Assays: Approved Guideline. 5th edn. CLSI document H21-A5, Wayne, PA: Clinical and Laboratory Standards Institute (CLSI); 2008. 21. Sixma JJ. Methods for platelet aggregation. Adv Exp Med Biol 1972;34:79–95. 22. Cattaneo M. Light transmission aggregometry and ATP release for the diagnostic assessment of platelet function. Semin Thromb Hemost 2009;35:158–67. 23. Thompson CB, Eaton KA, Princiotta SM, Rushin CA, Valeri CR. Size dependent platelet subpopulations: relationship of platelet volume to ultrastructure, enzymatic activity, and function. Br J Haematol 1982;50:509–19.
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24. Merolla M, Nardi MA, Berger JS. Centrifugation speed affects light transmission aggregometry. Int J Lab Hematol 2012;34:81–5. 25. Anstall HB, Hawkey CM. Observations on platelet clumping and related phenomena: a reappraisal and a clarification of terms. Transfusion 1962;2:44–51. 26. Kattlove HE, Alexander B. The effect of cold on platelets. I. Cold-induced platelet aggregation. Blood 1971;38:39–48. 27. Sixma JJ, Nijessen JG. Characteristics of platelet factor 3 release during ADPinduced aggregation. Comparison with 5 hydroxytryptamine release. Thromb Diath Haemorrh 1970;24:206–13. 28. Kattlove HE, Alexander B, White F. The effect of cold on platelets. II. Platelet function after short-term storage at cold temperatures. Blood 1972;40:688–96. 29. Mani H, Luxembourg B, Klaffling C, Erbe M, Lindhoff-Last E. Use of native or platelet count adjusted platelet rich plasma for platelet aggregation measurements. J Clin Pathol 2005;58:747–50. 30. Cattaneo M, Lecchi A, Zighetti ML, Lussana F. Platelet aggregation studies: autologous platelet-poor plasma inhibits platelet aggregation when added to platelet-rich plasma to normalize platelet count. Haematologica 2007;92:694–7. 31. Linnemann B, Schwonberg J, Mani H, Prochnow S, Lindhoff-Last E. Standardization of light transmittance aggregometry for monitoring antiplatelet therapy: an adjustment for platelet count is not necessary. J Thromb Haemost 2008;6:677–83.
ORIGINAL ARTICLE
INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY
Assessment of spontaneous platelet aggregation using laser light scattering in healthy subjects: an attempt to standardize S. SUZUKI* ,† , H. KUDO † , T. KOYAMA*
*Laboratory Molecular Genetics of Hematology, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan † Department of Clinical Laboratory Medicine, Faculty of Health Science Technology, Bunkyo Gakuin University, Bunkyo-ku, Tokyo, Japan Correspondence: Takatoshi Koyama, MD, PhD, Graduate School of Health Care Sciences, Tokyo Medical and Dental University 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. Tel./Fax: +81-3-5803-5882, E-mail: [email protected] doi:10.1111/ijlh.12208
Received 20 November 2013; accepted for publication 10 February 2014 Keywords Platelet, aggregation, spontaneous platelet aggregation (SPA), P-selectin, activated glycoprotein IIb/IIIa
S U M M A RY Introduction: When measuring platelet aggregation using laser light scattering, small aggregates forming without the addition of agonists may be observed. This event is called ‘spontaneous platelet aggregation (SPA)’. The platelet hyperactivity observed in arterial thrombotic diseases can be detected with relative ease by measuring SPA. Standardization is urgently needed because of differences between measurement conditions among various laboratories. Methods: We conducted a systematic study of factors that affect SPA measurement, compared SPA results to flow cytometry detection of surface antigens expressed on activated platelet membranes (P-selectin, activated glycoprotein IIb/IIIa), and determined conditions that yield stable measurements. Results and Conclusions: We evaluated results from 125 healthy volunteers and established conditions for a stable measurement of SPA. As the occurrence of SPA tended to increase with age, we determined conditions valid for subjects aged 20–60 years. Blood should be collected using a syringe, and the sample should be prepared after allowing the whole blood to rest for 30 min after collection. To isolate platelet-rich plasma, a 2-mL tube should be used and centrifuged at 150 g. The sample should be stored at room temperature, the platelet count of the sample should be (250 10) 9 109/L, and the measurement should be completed within 90 min of blood collection.
INTRODUCTION Platelets play an important role in hemostasis, and platelet hyperactivity is a factor in thrombus formation. Platelet hyperactivity is observed in arterial thrombotic diseases such as myocardial infarction and 676
cerebral infarction [1–4]. Therefore, understanding platelet hyperactivity is useful for diagnosis and treatment. If platelets are hyperactive, various changes occur in association with increased aggregatory reactivity and the release of contents of platelet granules in vivo. © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 676–685
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Therefore, historically, these items have been used as indicators to evaluate platelet hyperactivity. However, this method requires very expensive equipment, and the procedure itself is expensive, making it an uncommonly used method. One method of testing platelet function is the optical density (OD) method devised by Born [5]. This method was once the most commonly used method of undertaking a platelet-aggregation test; it is effective for diagnosing platelet dysfunction but has low sensitivity for detecting small aggregates of platelets and thus is difficult to use for evaluating platelet hyperactivity [6, 7]. However, particle-counting platelet aggregometers [8], which detect platelet aggregates using forward scattering of light with high-detection sensitivity and can even detect aggregates formed by only a few platelets, have made it possible to measure spontaneous platelet aggregation (SPA) that the OD method cannot detect. SPA is defined as small aggregates that form in vitro through stirring alone without the addition of agonists. Studies have reported that the number of these aggregates increases if platelets are hyperactive [9–12]. The laser light scattering (LS) method is based on the principle that the intensity of particle scattering is proportional to the square of the particle diameter and that the detected intensity of scattering can be used to obtain information regarding the number and size of aggregates in platelet-rich plasma (PRP). In this study, we used the LS method with a platelet aggregometer to explore simpler methods of detecting platelet hyperactivity by assessing platelet activity via detection of small platelet aggregates. However, it is known that measured values of SPA change depending on differences in conditions between blood collection and measurement. To enable wide application of measured values of SPA in clinical laboratories, they must have good precision and accuracy. This warrants standardization of conditions of blood collection and measurement [13–15]. Therefore, we systematically evaluated factors that influence SPA between blood collection and measurement to determine conditions that yield stable measurements. For confirmation, we compared SPA detection with flow cytometry (FCM) detection of activated glycoprotein (GP) IIb/IIIa (a fibrinogen receptor) and P-selectin (which reflects the release of © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 676–685
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a-granules). In addition, we attempted to determine conditions for measuring SPA that would be valid for 2 age groups of subjects in their 20 s and those aged ≥ 30 years.
M AT E R I A L S A N D M E T H O D S Subjects This study was approved by the ethics committee of Bunkyo Gakuin University (Tokyo, Japan). It was conducted on venous blood collected from 125 healthy, consenting students and faculty members (44 men and 81 women; 21–60 years). Blood from individuals taking drugs that could affect platelet aggregation was not used. Blood sampling Polypropyrene syringes and evacuated polyester tubes of 13.2φ 9 78 mm containing citrate were purchased from Terumo, Tokyo, Japan. Fasting blood samples were collected between 10:00 and 11:00 am to avoid diurnal variations in platelet aggregation [16]. Moreover, platelets can be activated by blood collection, and there can be individual differences in collection methods. Therefore, two experienced technicians collected all blood samples. Blood with air bubbles and blood that was difficult to collect was not used; only blood that was readily collected within 30 s of puncture was used. 12 mL of blood was collected from the median antebrachial vein using a tourniquet and a syringe with a 21-G needle. Collection was completed within 30 s of puncture. When SPA was measured, the initial 2 mL of blood collected was discarded, and the remaining blood was used. After the initial 2 mL of blood was discarded, the rest was divided into 2- and 5-mL tube without or with a top seal in a balanced order (i.e., into an evacuated tube first in some subjects and second in other subjects). Procedure and methods of SPA measurement
Preparation of measurement samples One unit of 109 mM sodium citrate had been added per nine units of blood. The tube was gently inverted
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three times and the blood left to rest at 25 °C for 30 min. PRP was isolated by centrifugation of blood at 150 g for 10 min at 25 °C. The remaining blood was centrifuged at 2000 g for an additional 10 min at 25 °C to isolate platelet-poor plasma (PPP). PPP was used to dilute PRP from the same subject to prepare a sample with a platelet count of (250 10) 9 109/L. A multi-item automatic blood cell analyzer (XS-1000i; Sysmex, Tokyo, Japan) was used to calculate the platelet count. Polypropyrene tubes (12φ 9 75 mm) for flow cytometric analysis (Beckman Coulter, Pasadena, CA, USA) were used during the preparation and handling of PRP.
SPA measurement method Spontaneous platelet aggregation was measured using a laser-scattering particle-counting platelet aggregometer (PA-20 of 2 channels and PA-200 of 4 channels; Kowa, Tokyo, Japan). To compare data, we ensured that there was no difference between channels of the device. The sample for measurement was dispensed into a cuvette with a stirring bar. After preheating the sample at 37 °C for 2 min, aggregation was measured for 10 min while only stirring at 2.43 g and without adding an agonist. SPA was evaluated using the maximum value of total light scattering intensity by small aggregates for a 10 min (SMax); SMax ≥ 2.0E4 mV 9 count was considered SPA positive (Figure 1). Other platelet-detection methods in comparison with SPA Rates of expression of activated GPIIb/IIIa and P-selectin, which are expressed on the surface of activated platelet membranes, were measured by FCM using monoclonal PAC-1 antibodies and anti-CD62P antibodies [17]. The flow cytometer used was an Epics XL (Beckman Coulter). A testing center (SRL, Tokyo, Japan) was commissioned to measure levels of bthromboglobulin and platelet factor 4, which are markers of platelet activation and indicators of the release reaction [18], using Asserachrom PF-4 and Asserachrom b-TG (Roche Diagnostics, Tokyo, Japan), respectively. Adenosine diphosphate (ADP) aggregation at a final concentration of 2 lM and collagen aggregation at a final concentration of 1 lg/mL were
Figure 1. SPA-positive record of time-dependent formation of platelet aggregates measured by laser light scattering (LS) method and optical density (OD) method in the absence of aggregation inducers as compared with ADP-induced traces. The total LS intensity reflected by small aggregates containing four to nine platelets increased slightly in a timedependent manner (-(S)-, blue curve), and there was virtually no change in the total LS intensity reflected by medium containing 10 to 99 platelets (-(M)-, green curve) and large containing over 100 platelets (-(L)-, red curve) aggregates. No increase of OD method (-(T)-) was observed in subjects. (1) We measured SPA as the maximum value of total LS intensity by small aggregates for a 10 min (SMax); SMax ≥ 2.0E4 was considered SPA-positive. This case showed 12E4 of SMax. (2) shows traces of 2 lM of ADP-induced platelet aggregation.
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measured using the PA-20 and PA-200 systems using the OD method.
indication for platelet aggregates and large and immature platelets.
Items assessed
Thirty-minute sample resting period before measurement
Experiments presented below were performed using the above-mentioned blood sampling method and SPA measurement procedures. Only assessed items were changed; all other conditions were the same. To avoid the influence of time, SPA measurements were undertaken simultaneously. To avoid effects of platelet activation and pH changes, all experiments were performed in a room maintained at 25 1 °C, and samples were stored in tightly sealed polypropylene test tubes.
Method of blood collection and anticoagulation Effects of the blood collection method (syringe or vacuum tube containing citrate) and the concentration of the anticoagulant sodium citrate (106 or 109 mM) on SPA were assessed using blood samples collected from the same subject by measuring and comparing SPA as well as surface antigen expression. Osmotic pressure of PRP was measured with Osmo STAD OM-6040 (Arkray, Kyoto, Japan).
The stability of allowing whole blood to rest for 30 min immediately after collection was compared with the stability of leaving a prepared measurement sample of blood from the same subject to rest for 30 min based on SPA and surface antigen expression.
Storage temperature of samples After storing the prepared measurement samples from the same subject for 30 min at 4, 25, or 30 °C, SPA was simultaneously measured to evaluate effects of storage temperature on SPA.
Platelet count Measurement samples were prepared with four platelet concentrations (100, 250, 300, 400) 9 109/L with blood from the same subject. SPA was simultaneously measured to evaluate the effects of the platelet count on SPA.
Centrifugal force Blood from the same subject was dispensed into two blood-collection tubes of the same size (2 and 5 mL) that contained 0.2 and 0.5 mL of the anticoagulant. Tubes were simultaneously centrifuged at 150 g for 10 min at 25 °C. Measurement samples were prepared, and SPA was simultaneously measured. Effects of centrifugal force while separating PRP were examined using SPA along with the platelet large-cell ratio (P-LCR), mean platelet volume (MPV), and platelet distribution width (PDW) of the measurement sample. The relative centrifugal force (RCF) at this time was calculated as the average of the centrifugation force (g) applied to the bottom of the test tube and that applied to the liquid surface. The P-LCR, MPV, and PDW were measured using the XS-1000i multi-item automatic blood cell analyzer. The P-LCR indicates the percentage of large platelets with a volume > 12 fL. An increase in the parameter may be an
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Time-dependent changes Measurement samples prepared from the same subject were measured 60, 90, 120, and 180 min after collection to examine the effect of the time elapsed between collection and measurement on SPA.
Age-related differences in SPA Using the same method of blood collection, SPA values from samples that had been prepared and measured were examined according to age group. Statistical analyses SPSS ver18 (IBM, Tokyo, Japan) was used for calculations. Correlation analyses were performed using Spearman’s correlation. Tests of significance were conducted using repeated-measures ANOVA and Wilcoxon
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t-tests. P < 0.05 was considered significant. The data were shown with mean SEM.
R E S U LT S The correlation between SPA and other methods of detection of platelet activation is presented in Table 1. Correlations with SPA were determined for CD62P and PAC-1; correlation coefficients were 0.440 and 0.705, respectively. SPA values for the syringe method of blood collection were significantly lower than those of the vacuum-tube method, and the expression of P-selectin and activated GPIIb/IIIa was lower, although not significantly so (Table 2-1). No significant difference in SPA values was observed between 106 and 109 mM concentrations of the anticoagulant sodium citrate, which are both commonly used. pH levels of these samples at the time of measurement were 7.91 and 7.89, respectively, and there was no significant difference in osmotic pressure (Table 2-2). In the experiment focusing on effects of centrifugal force on SPA, RCF applied to 2- and 5-mL test tubes was 139 and 124 g, respectively, and SPA was significantly higher for the 2-mL tube, which experienced stronger RCF. In contrast, the P-LCR, MPV, and PDW were significantly higher for the 5-mL tube, which experienced weaker RCF (Table 2-3). In the experiment with the 30-min resting period between blood collection and measurement, allowing the blood to rest as whole blood resulted in a significantly lower SPA than preparing the sample by isolating PRP before the resting period (Table 2-4). Moreover, the expression of activated GPIIb/IIIa was
Table 1. Correlation between SPA and other methods of detection of platelet activation
Platelet aggregation Platelet activation markers
Class
n
r
P value
ADP (%) Collagen (%) b-TG (ng/mL) PF-4 (ng/mL) CD62P expression (%) PAC-1 expression (%)
68 68 35 35 45
0.277 0.172 0.199 0.370 0.440
0.022 0.162 0.251 0.029 0.002
45 0.705 0.000
Table 2. Effects of (1) blood collection method on platelet activation (2) anticoagulant sodium citrate on SPA (3) Centrifugal force on platelet activation (4) Platelet activation that 30-min resting period gives (1)* Vacuum tube
P value
Collection method
Syringe
SPA (SMax E4) CD62P expression (%) PAC-1 expression (%)
2.95 0.75 3.56 0.82 1.26 0.17 1.40 0.21
0.028 0.611
1.35 0.29 1.71 0.28
0.120
(2)† Concentration pH Osmotic pressure (mOsm/kg) SPA (SMax E4)
106 mM (3.13%)
109 mM (3.20%)
P value
7.91 0.05 7.89 0.06 0.206 283.3 1.69 283.3 1.51 0.922 4.41 1.16
4.59 1.05 0.167
(3)‡ Blood-collection tube RCF (9g)
2 mL
5 mL
139
129
SPA (SMax E4) P-LCR (%) MPV (fL) PDW (fL)
1.88 14.39 8.83 9.13
0.41 1.43 0.27 1.16 20.79 1.51 0.15 9.89 0.34 0.29 10.39 0.34
P value 0.034 0.000 0.002 0.000
(4)§ Prepared sample SPA (SMax E4) CD62P expression (%) PAC-1 expression (%)
Whole blood
P value
4.97 0.75
4.04 0.60 0.037
6.56 0.67
5.05 1.01 0.099
33.35 2.42
29.41 1.87 0.028
PLC-R, platelet large-cell ratio; MPV, mean platelet volume; PDW; platelet distribution width. *n = 6. †n = 8. ‡n = 16. §n = 12.
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significantly lower, and the expression of P-selectin was lower, but not significantly so. Platelets with a low SPA value were not affected by the storage temperature, but platelets that were
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SPA positive and moderately active revealed higher SPA under lower storage temperatures (Figure 2-1). In the experiment focusing on the effect of platelet count on SPA, higher platelet counts tended to be
Figure 2. Effects of measurement condition on SPA. Experiments were performed using the same method of blood sampling and SPA measurement procedures. Only assessed items were changed; all other conditions were the same. SPA values from samples were examined according to 2 age group (subjects in their 20s and subjects aged ≥ 30 years). The dark gray column expresses SPA values from subjects in their 20s, and the bright gray column expresses subjects aged ≥ 30 years, and the line graph expresses overall mean. Subjects aged ≥ 30 years were comprised of 1-2 subjects chosen from every age group. (2-1): Effect of storage temperature on SPA. After storing prepared measurement samples from the same subject for 30 min at 4, 25, or 30 °C. Subjects in their 20s were 7 peoples and subjects aged ≥ 30 years were 7 peoples. No significant difference was observed between SPA at the platelet storage temperature at 25 °C, and platelet storage temperature at 30 °C, with respect to overall mean SPA and SPA of subjects in their 20s. But a significant difference was observed in subjects aged ≥ 30 years. This suggests that sample storage at room temperature (25 °C) is suitable for both age groups. (2-2): Effect of platelet count on SPA. Measurement samples were prepared with four platelet concentrations (100, 250, 300, 400) 9 109/L with blood from the same subject. Subjects in their 20s were 6 peoples and subjects aged ≥ 30 years were 6 peoples. SPA significantly changed with the platelet count. No significant difference was observed between SPA at a platelet concentration of 250 9 109/L (which is most reactive and has the highest SPA), and platelet concentrations of 250–300 9 109/L with respect to overall mean SPA and SPA of subjects aged ≥ 30 years, but a significant difference was observed in subjects in their 20s. This suggests that a concentration of 250 9 109/L is suitable for both age groups. (2-3): Effect of time-dependent changes on SPA. Measurement samples prepared from the same subject were measured 60, 90, 120, and 180 min after collection. Subjects in their 20s were 5 peoples and subjects aged ≥30 years were 5 peoples. SPA increased significantly with longer time intervals regardless of age. SMax of ≥2.0E4 was considered to indicate SPA. Blood samples revealed only a normal SPA range for healthy individuals (2.0E4) ≤90 min after collection. This result suggests that SPA measurements should be completed ≤90 min of blood collection.
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associated with higher SPA regardless of age of the subject, and a significant difference was observed for all intervals except 250–300 9 109/L (Figure 2-2). In addition, SPA increased significantly with longer time intervals in the experiment investigating time-dependent changes until measurement of the sample (Figure 2-3). All subjects were healthy, but some samples were SPA positive (scattering intensity of 2.0E4) when measured after >90 min had elapsed since sampling. Spontaneous platelet aggregation values of 50 healthy subjects measured are presented according to age in Figure 3. The SPA-positive rate was calculated for 2 age groups of subjects in their 20s and those aged ≥ 30 years. The positive rate tended to increase with age.
DISCUSSION Activated GPIIb/IIIa and P-selectin were used as points of comparison for SPA in the present study because they significantly correlated with SPA, and are thus useful for detecting platelet activation (hyperactivity).
Figure 3. Age-related differences in SPA. SPA values from samples of 50 healthy subjects were examined according to age group. Subjects in their 20s were 10 peoples and those aged ≥ 30 years were 40 peoples. SPA-positive (SMax ≥ 2.0E4) rate was calculated for each age group, which tended to increase with age. SPA-positive rate of subjects in their 20s were 20% (2/10) and those aged ≥ 30 years were 50% (20/40). Even if the positive rate of subjects in their 20s calculated in 45 subjects, it was 20% (9/45).
It is recommended that a 19-G needle be used without a tourniquet when collecting blood for platelet-aggregation tests. The normal practice is to use a tourniquet for a minimal amount of time in order to insert the needle and then to release the pressure during blood collection to avoid hemoconcentration and activation. As it is often difficult to take blood from a Japanese female using a 19-G needle, we collected blood using a tourniquet and a 21-G needle, the most commonly used needle. We compared the syringe and vacuum-tube methods of blood collection and determined that collecting samples using a syringe affected platelet activation significantly less often compared with collecting samples with a vacuum tube, which suggests that using a syringe is a better method of blood collection. When FCM was used, the syringe method of blood collection revealed decreased expression of activated GPIIb/IIIa and P-selectin compared with that in the vacuum-tube method. This increase in SPA probably resulted because of moderate activation of platelets induced by the strong force of blood that is sucked in by the vacuum hitting the test tube wall. The World Health Organization (WHO) and Clinical and Laboratory Standards Institute (CLSI) recommend that anticoagulant sodium citrate concentrations of 3.2% (which is isotonic with blood) should be used in coagulation tests [19, 20]. However, the desirable concentration for platelet-aggregation tests is not clear. Therefore, we examined two commonly used sodium citrate concentrations (106 and 109 mM) but found no significant difference between them. A few studies [21, 22] have suggested that platelet aggregation is affected by pH, but minor changes observed in the present study were not strong enough to affect SPA. Centrifugation must be performed when conducting a platelet-aggregation test. We examined two commonly used volumes of blood mixed with anticoagulant in a collection tube (2 and 5 mL). On investigating the effect that RCF, which was used to obtain PRP, had on SPA, we determined that SPA was higher in tubes with 2 mL blood, which have a higher RCF applied to them. This result contradicts that of studies [23, 24] suggesting that a high RCF removes highly active large platelets and therefore yields low aggregation. However, the platelet large-cell ratio and the mean platelet volume were higher for 5-mL tubes, © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 676–685
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which had a lower RCF applied to them. The reason why these tubes revealed lower SPA may have been that the 2-mL tubes, with high RCF applied to them, may have experienced a large shearing stress. Collecting 5 mL blood not only yields a lower PRP volume and a larger platelet count in PRP, thereby making sample preparation for measurement more time-consuming, but also causes other inconveniences such as platelet activation during sample preparation. However, collecting 2 mL blood is convenient because it allows for a smaller amount of blood to be collected because of the large amount of PRP that can be obtained, and the platelet concentration in PRP is close to the concentration of 250 9 109/L that is to be prepared, which makes it easy to prepare samples for measurement and prevents platelet activation. Standardization of centrifugation conditions is important for standardization of the entire process, but the volume of blood collected and the size of blood-collection tube must also be taken into account. Our results suggest that in clinical laboratories, 2 mL blood should be collected and spun at 150 g. We used SPA and FCM to determine whether blood should be left to rest as whole blood or as a prepared sample during the 30 min between blood collection and measurement and found that leaving blood as whole blood yielded good SPA results. The reason for this may be that the ‘double load’ placed on blood by collection and producing PRP through centrifugation has a stronger effect on platelets and that subjecting blood to the load of PRP isolation after the load from blood collection has subsided is a more suitable method. The fact that the expression of activated GPIIb/IIIa using FCM was higher in samples that were immediately prepared also suggests that this double load induced further activation, which could have caused platelets to sensitively react to even slight stimuli, thereby causing SPA to increase. This result suggests that it is best to let the blood rest as whole blood immediately after collection. This result will be very helpful for busy clinical facilities that process a large number of blood samples because samples can be processed in batches, and there is no need to centrifuge individual samples immediately to prepare PRP. Studies [25, 26] have reported that platelet aggregation increases at low storage temperatures. We examined the effects of storage temperature on plate© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 676–685
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let activation during the 30-min resting period. We observed that preheating platelets of subjects in their 20s at 37 °C for 2 min just before platelet aggregation measurement negated any effect of storage temperature, but that storing platelets of subjects aged ≥ 30 years at 4 °C caused SPA to significantly increase and at 30 °C caused SPA to significantly decrease. Therefore, storing platelets at low temperatures controlled dissociation and enhanced aggregation [27, 28], but preheating platelets of subjects in their 20s at 37 °C for 2 min dissociated aggregates and thus did not affect SPA. In contrast, platelets of older subjects were in a moderately active state and thus may not have dissociated aggregates as easily. This result suggests that taking the age and pathological condition of patients into account, samples should be stored at room temperature (25 °C) during the 30min resting period when conducting a test. We used our room temperature of 25 °C, as in Japan, a hematological laboratory room is generally linked to outpatient rooms in a hospital and the temperature in the hospital is regulated to 25 °C. Resting for 30 min seems to be necessary to escape from stimulus of blood collection procedure and to be convenient for operators to prepare for the SPA measurement. Resting for 2 h may not be a problem for measurement of agonist-induced platelet aggregation, but as indicated in Figure 2-3, more than 1 h after collection, SPA is induced. As SPA should be measured concentratedly with reservation, hospital transport systems may not be suitable for SPA measurement. We examined effects of the platelet count of the measurement sample on SPA and found that SPA significantly changed with the platelet count. No significant difference was observed between SPA at a platelet concentration of 250 9 109/L, which is most reactive and has the highest SPA, and platelet concentrations of 250–300 9 109/L with respect to overall mean SPA and SPA of subjects aged ≥ 30 years, but a significant difference was observed in subjects in their 20s. This suggests that a concentration of 250 9 109/L is suitable for both age groups, and this is the easiest concentration to prepare in clinical settings (it is difficult to prepare a concentration of 300 9 109/L from a patient with a low platelet count). The probable reason why a difference was observed between subjects in their 20s and those aged ≥ 30 years may be that platelets of subjects in their 20s reacted sensitively to
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agitation by the stirrer during measurement depending on the platelet count, but that platelets that are already moderately activated react similarly and are not affected by minor changes in the platelet count if SPA is high. Studies [29–31] have shown that it is not necessary to adjust the platelet count of measurement samples when using conventional methods of measuring platelet aggregation (e.g., ADP and collagen aggregation). However, SPA reacts sensitively to platelet count because it measures aggregation by detecting aggregates that are too small to affect optical density, and several samples have a PRP platelet count outside the range of 250–300 9 109/L, which is why platelet count should be adjusted when using this method. We evaluated changes in SPA based on the time elapsed from blood collection to measurement. We observed that SPA increased significantly with longer time intervals regardless of age. SMax of ≥2.0E4 was considered to indicate SPA. Blood samples revealed only a normal SPA range for healthy individuals (2.0E4) ≤90 min after collection. This result suggests that SPA measurements should be completed ≤90 min of blood collection. When we measured SPA under conditions described above, we determined a larger prevalence of SPA positives (SMax ≥ 2.0E4) in subjects aged ≥ 30 years compared with subjects in their 20s, although all subjects were healthy. Only 20% subjects in their 20s were positive for SPA, whereas 50% subjects aged ≥ 30 years were positive. This age-related increase in SPA suggests the following: (i) age must be taken into account when setting standard values, (ii) platelets become unstable and increasingly reactive with age although they are
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AC K N OW L E D G E M E N T S We are very grateful to Dr Shinobu Sakamoto for appropriate support and Kumiko Yanagisawa for assisting with our experiments.
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