Evaluation of Automated Platelet Aggregation Test
Evaluation of Automated Platelet Aggregation Test
Evaluation of Automated Platelet Aggregation Test
원저
Lab Med Online
Vol. 10, No. 2: 137-143, April 2020 진단혈액학
https://doi.org/10.3343/lmo.2020.10.2.137
https://crossmark-cdn.crossref.org/widget/v2.0/logos/CROSSMARK_Color_square.svg 2017-03-16
Background: The platelet aggregation test is widely used to measure antiplatelet therapy response and to detect platelet function disorders.
CS-5100 (Sysmex Co., Japan) is a recently introduced coagulation analyzer that can also measure platelet aggregation. We evaluated the perfor-
mance of CS-5100 in the platelet aggregation test for use in clinical laboratories.
Methods: We investigated the precision, stability, dilution test, and correlation of CS-5100 with a traditional aggregometer. Precision was tested
using normal and patient samples. Stability was assessed over 5 different time points for 8 hours. The dilution test was performed with normal
samples using ADP agonists. We tested correlations between the results of Chrono-log aggregometer (Chrono-Log Co., USA) and CS-5100 using
10 samples with 5 agonists each. We also calculated the reference range of 5 agonists using 22-30 normal samples.
Results: The coefficients of variation using normal samples were 7.45% and 3.27% for ADP and arachidonic acid, respectively. Stability was
maintained for up to 2 hours in most samples. Dilution tests showed similar results until reaching a dilution factor of 2. Strong correlations of the
results between Chrono-log and CS-5100 were found, except for ristocetin. The reference ranges of 5 reagents in CS-5100 were similar to those
obtained with the Chrono-log aggregometer.
Conclusions: The performance of CS-5100 in platelet aggregation tests showed reliable results compared to a traditional aggregometer. CS-
5100 can perform coagulation test and platelet aggregation test, simultaneously. Thus, CS-5100 can enable cost saving and reduce turn-around-
time by reducing the inspection time.
Key Words: CS-5100, Platelet aggregation test, Coagulation instrument, ADP, Arachidonic acid
saves time. It uses several agonists for the platelet aggregation 3. Reagents
test, including ADP, arachidonic acid, collagen, epinephrine, and CHRONO-PAR Reagents (BioTop Medical, Leiden, Netherlands)
ristocetin [7]. Blood sampling and centrifugation are performed were used for the platelet aggregation tests and included ADP, ar-
manually in CS-5100; however, platelet rich plasma (PRP) and achidonic acid, collagen, epinephrine, ristocetin, and thrombin.
platelet poor plasma (PPP) are dispensed automatically, and ago- Among these, five reagents were used for platelet aggregation tests,
nist addition is also automatic. We evaluated the performance of including ADP, arachidonic acid, collagen, epinephrine and risto-
CS-5100 in evaluating platelet aggregation for use in clinical labo- cetin. The final concentrations of the reagents were determined
ratories. We measured the precision, stability, dilution test, corre- according to the guidelines of the International Society of Throm-
lation of the two instruments, and the reference range of five re- bosis and Hemostasis (ISTH) [8]. The concentrations of ADP, ara-
agents. chidonic acid, collagen, epinephrine, and ristocetin were 2 μM,
1.0 mM, 2 μg/mL, 5 μM, and 1.2 mg/mL, respectively.
MATERIALS AND METHODS
4. Precision
1. Instruments Normal and patient samples were also tested to evaluate preci-
The CS-5100 is used in our laboratory for blood coagulation sion. The test was repeated five times within one day. The reagents
tests. It also can be used to measure platelet aggregation. It per- used were ADP and arachidonic acid. Mean values, SD and CVs
forms the platelet aggregation tests using light transmission ag- were calculated. The test was performed following the Clinical
gregometry. After preparing the sample and reagent, the instru- and Laboratory Standard Institute (CLSI) guidelines EP5-A3 [9].
ment analyzes the PRP and PPP samples and then automatically
calculates the aggregation %. Aggregation % is calculated using 5. Stability
PRP results, PPP results, and the degree of absorbance. The range Stability was assessed by measuring normal and patient sam-
of the results is from -25% to 100%. A Chrono-log aggregometer ples at five different times. The test was performed immediately
(Chrono-Log Co., Havertown, PA, USA) was used for the correla- and at 1 hour, 2 hours, 4 hours, and 8 hours after phlebotomy us-
tion test. ing ADP and arachidonic acid. The results of stability were ana-
lyzed statistically by Mann-Whitney test.
2. Samples
This study was approved by the institutional review board of 6. Dilution tests
Dong-A University Hospital (Busan, Korea). The evaluated blood Dilution tests were performed to measure the effect of platelet
samples were collected in 3.2% sodium citrate tubes (Greiner Bio- counts. Dilution tests were only performed with normal samples.
One, Frickenhausen, Germany) and mixed gently. To obtain PRP The tests were performed with several dilution factors of 1, 2, 4, 8,
samples, samples were centrifuged at 200 g for 10 minutes at room 16, 32, 64, and 128. The agonists ADP and arachidonic acid were
temperature. The resultant PRP samples were transferred to a plas- used to analyze the dilution tests. We also measured the platelet
tic tube using a plastic pipette. To obtain PPP samples, samples counts.
were centrifuged at 1,500 g for 15 minutes at room temperature.
The resultant PPP samples were also transferred to a plastic tube. 7. Correlation tests
9
Platelet counts in PPP samples were less than 10 × 10 /L. Normal Correlation tests were performed following the CLSI guidelines
samples were obtained from a health screening center, and pa- EP9-A3 [10]. Correlations between the results of Chrono-log ag-
tient samples were residual samples obtained from patients with gregometer and CS-5100 were assessed using 10 samples with five
cerebrovascular disease such as cerebral infarction, cerebral hem- reagents (ADP, arachidonic acid, collagen, epinephrine, ristoce-
orrhage, and so on. tin) each. The concentrations of the reagents used in CS-5100 were
determined using ISTH guidelines [8]. Correlation between the
CS-5100 and Chrono-log aggregometer was analyzed using Mi-
(67.7% (42.0–71.5) vs. 26.3% (-23.0–58.3); P = 0.1266). In patient ar- and arachidonic samples showed similar results until reaching a
achidonic acid samples, the stability results were too low to be dilution factor of 2.
analyzed.
4. Correlation with a traditional aggregometer
3. Dilution tests The correlation test results are displayed in Fig. 1. In ADP and
Dilution test results are also displayed in Table 3. The initial plate- arachidonic acid treatment samples, the results of the CS-5100 and
let count of the normal samples was 377,000/μL. Tests using ADP Chrono-log aggregometer were strongly correlated (R = 0.955, R =
0.996, respectively) [12, 13]. The results of collagen and epineph-
Table 3. Dilution test using normal ADP samples rine samples were correlated, but not very strongly (R = 0.881, R =
Dilution Platelet count ADP (aggrega- Arachidonic acid 0.895, respectively) [12, 13]. Finally, ristocetin addition revealed a
Sample No.
factor (/μL) tion %) (aggregation %)
modest correlation result (R = 0.586) [12, 13].
1 1 377,000 85.6 90.0
2 1/2 193,000 89.9 91.4
3 1/4 98,000 85.6 7.2 5. Reference ranges of the CS-5100 platelet aggregation
4 1/8 51,000 6.9 2.5
test
5 1/16 24,000 -15.1 -3.1
6 1/32 12,000 -8.3 5.6 The test results are shown in Table 4. All tests using five ago-
7 1/64 6,000 -3.8 0.0 nists showed normal distribution; the reference range was calcu-
8 1/128 3,000 -3.1 0.0
lated as mean ± 2SD. The reference ranges were 59.3-105.2% for
80 y=1.4721x-27.012 80 y=1.0174x+2.7539
R=0.955 R=0.996
60 60
40 40
20 20
0 0
20 40 60 80 100 20 40 60 80 100
Aggregometer aggregation (%) A Aggregometer aggregation (%) B
Collagen Epinephrine
100 100
CS-5100 aggregation (%)
80 y=1.1053x+2.2347 80 y=0.8535x+10.298
R=0.881 R=0.895
60 60
40 40
20 20
0 0
20 40 60 80 100 20 40 60 80 100
Aggregometer aggregation (%) C Aggregometer aggregation (%) D
Ristopcetin
100
CS-5100 aggregation (%)
80 y=0.4118x+40.642
R=0.586
60
40
20
0 Fig. 1. Correlation of the test results between Chrono-log aggregom-
20 40 60 80 100 eter and CS-5100 aggregation assay. (A) ADP, (B) Arachidonic acid, (C)
Aggregometer aggregation (%) E Collagen, (D) Epinephrine, and (E) Ristocetin.
Table 4. Reference range of CS-5100 automated platelet aggregation 200,000/μL, the results of aggregation were reliable.
assay
In a comparison test between the CS-5100 and Chrono-log ag-
Agonist N Mean (%) SD Mean ± 2SD (%) gregometer, the results using four reagents except ristocetin showed
ADP 27 82.3 11.5 59.3-105.2
strong correlations. Use of ADP and arachidonic acid showed strong
Arachidonic acid 22 82.6 9.7 63.2-102.0
Collagen 30 88.3 7.2 74.0-102.7 correlations (R = 0.955, R = 0.996, respectively) [12, 13]. Use of col-
Epinephrine 30 88.6 7.9 72.8-104.3 lagen and epinephrine showed relatively weaker correlation (R =
Ristocetin 30 83.8 7.0 69.9-97.8 0.881, R = 0.895, respectively) [12, 13]. In contrast, the results of ris-
tocetin showed a modest correlation (R = 0.586) [12, 13]. These re-
ADP, 63.2-102.0% for arachidonic acid, 74.0-102.7% for collagen, sults are likely caused because of not using a dedicated reagent or
72.8-104.3% for epinephrine, and 69.9-97.8% for ristocetin. using the wrong concentration of the reagent obtained from the
manufacturer.
DISCUSSION The reference ranges of CS-5100 were not significantly different
from those of traditional aggregometers [14, 15]. In reference range
Platelet aggregation tests are being increasingly used because tests, using ADP and arachidonic acid provide wider ranges than
of their usefulness in diagnosis and to monitor therapeutic response. with the other three reagents. However, the numbers of samples
The fully automated CS-5100 coagulation and platelet aggregation used for the assay was small; thus, additional samples are required
analyzer is thus expected to be more useful in hospitals and labo- for further performance evaluation.
ratories. The results of the CS-5100 platelet aggregation test are similar to
The CS-5100 analyzer yielded acceptable precision with normal other results reported using CS-2100i [16, 17]. However, our analy-
samples using both ADP and arachidonic acid. However, the re- sis has some limitations. First, because of a small number of sam-
sults of patient samples were not acceptable. The number of sam- ples, the SD and CV results were not good. We only tested ADP
ples was only five and the aggregation test results were too low; and arachidonic acid in precision, stability, and dilution tests. The
thus, the CV results of patient samples were very high and not ac- precision tests were performed only five times, and so, their re-
ceptable. Therefore, more samples and additional reagents are re- sults might not be reliable. Second, the reagents were not dedicated
quired for further performance evaluation. for CS-5100, and were also used for the Chrono-log aggregometer.
The CS-5100 provided good stability in both normal and pa- Therefore, the results of some tests were not acceptable. Further,
tient samples using ADP and arachidonic acid. Results of ADP in additional samples are needed to evaluate accuracy. Dedicated re-
normal samples were adequate until 2 hours after phlebotomy. agents for CS-5100 are needed in further evaluations. Third, only
Results using arachidonic acid in normal samples were acceptable samples from patients with some cerebrovascular diseases were
until 4 hours after phlebotomy. In patient samples, results using used; thus, additional tests are needed with samples of patients
ADP in samples were good until 4 hours after phlebotomy. Re- with coagulation disorders such as Glanzmann’s thrombasthenia,
sults using arachidonic acid in patient samples were all similar af- Bernard-Soulier Syndrome, and so on.
ter phlebotomy, but were very poor and could not be analyzed. Despite some limitations, the performance of the CS-5100 plate-
Thus, more samples and additional evaluation are required for ac- let aggregation test was reliable in terms of precision, stability, and
curate performance measurements. These results indicate that dilution tests. The CS-5100 showed results comparable to those of
when using CS-5100, tests should be performed within 2 hours af- a traditional aggregometer and has many advantages as it can ex-
ter phlebotomy. ecute coagulation tests and platelet aggregation tests without an
Tests using ADP and arachidonic acid are most frequently used additional aggregometer. It can also reduce the complexity of for-
in the clinical laboratory, and so, dilution tests were performed mer aggregometers and manual tests. Therefore, we expect that
with ADP and arachidonic acid agonists. In dilution tests, the re- the use of CS-5100 will be profitable in many ways to save time
sults using ADP and arachidonic samples were adequate until 2 × and increase convenience.
dilution. Thus, if the platelet counts of samples are more than
16. Ling LQ, Liao J, Niu Q, Wang X, Jia J, Zuo CH, et al. Evaluation of an tion of routine light transmission platelet aggregation. Int J Lab Hema-
automated light transmission aggregometry. Platelets 2017;28:712-9. tol 2014;36:431-8.
17. Lawrie AS, Kobayashi K, Lane PJ, Mackie IJ, Machin SJ. The automa-