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Vol. 10, No. 2: 137-143, April 2020 진단혈액학
https://doi.org/10.3343/lmo.2020.10.2.137
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Sysmex CS-5100의 자동화 혈소판 응집검사 평가

Evaluation of Automated Platelet Aggregation Test Using a Sysmex CS-5100 Analyzer
안규대1·정인화1·임현호2·우광숙1·김경희1·김정만3·한진영1
Gyu-Dae An, M.D.1, In-Hwa Jeong, M.D.1, Hyeon-Ho Lim, M.D.2, Kwang-Sook Woo, M.D.1, Kyeong-Hee Kim, M.D.1,
Jeong-Man Kim, M.D.3, Jin-Yeong Han, M.D.1
동아대학교 의과대학 진단검사의학과1, 울산병원 진단검사의학과2, 씨젠의료재단3
Department of Laboratory Medicine1, Dong-A University College of Medicine, Busan; Department of Laboratory Medicine2, Ulsan Medical
Center, Ulsan; Seegene Medical Foundation3, Busan, Korea

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

INTRODUCTION associated with various diseases such as immune thrombocyto-

penic purpura, thrombotic thrombocytopenic purpura, hemolytic
Platelets are blood cells present in peripheral blood. They are uremic syndrome, essential thrombocythemia, Bernard-Soulier
responsible for primary hemostasis through adhesion and coagu- Syndrome, and Glanzmann’s thrombasthenia due to numerical
lation [1]. They are mainly produced in the bone marrow and are abnormalities and dysfunction [2, 3]. Various tests are performed
in hospitals to measure platelet function. Among these, the plate-
Corresponding author: Jin-Yeong Han, M.D., Ph.D.
https://orcid.org/0000-0003-0280-2739
let aggregation test is widely used to measure antiplatelet therapy
Department of Laboratory Medicine, Dong-A University College of Medicine, responses and to detect congenital and acquired platelet function
26 Daesingongwon-ro, Seo-gu, Busan 49201, Korea
Tel: +82-51-240-5323, Fax: +82-51-255-9366, E-mail: [email protected] disorders [4, 5]. Light transmission aggregometry is the most com-
mon platelet aggregation test [6]. Conventional analyzers are usu-
Received: March 5, 2019
Revision received: August 14, 2019 ally semi-automated with result analysis and aggregation detec-
Accepted: September 5, 2019 tion performed automatically, whereas sample dispensing and ag-
This article is available from https://www.labmedonline.org onist addition performed manually.
2020, Laboratory Medicine Online The CS-5100 (Sysmex Co., Kobe, Japan) is an automated blood
This is an Open Access article distributed under the terms of the Creative Commons
Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) coagulation analyzer that can also measure platelet aggregation.
which permits unrestricted non-commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited. The CS-5100 is a fully automated analyzer that is convenient and

eISSN 2093-6338 www.labmedonline.org 137

 안규대 외: Platelet Aggregation Test Using CS-5100

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-

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안규대 외: Platelet Aggregation Test Using CS-5100

crosoft Excel 2010 (Microsoft Co., Redmond, WA, USA). 2. Stability

The results of stability testing are shown in Table 2. In normal
8. Reference ranges ADP samples, stability at 4 hours after phlebotomy was signifi-
The reference ranges of the five reagents were investigated us- cantly low (92.7% (88.9–97.6) vs. 63.3% (40.9–86.6); P = 0.0495). In
ing 22-30 normal samples based on the calculated mean ± 2SD of normal arachidonic acid samples, stability at 8 h after phlebotomy
all test samples. Tests to measure reference ranges were performed was significantly low (85.9% (85.7–93.7) vs. 3.4% (1.9–77.8);
following the CLSI guidelines EP28-A3C [11]. The numbers of sam- P = 0.0495). In patient ADP samples, stability at 8 hours after phle-
ples used for the assay were 27, 22, 30, 30, and 30 for ADP, arachi- botomy was low, but this result was not statistically significant
donic acid, collagen, epinephrine, and ristocetin, respectively.
Table 1. Within day precision of CS-5100 platelet aggregation analysis
Parameter Normal (aggregation %) Abnormal (aggregation %)
9. Statistical analyses
ADP
All statistical analyses were performed with Microsoft Excel 2010
1 69.7 17.8
(Microsoft Co.) and MedCalc software v16.4.3 for Windows (Med- 2 81.4 16.4
Calc, Ostend, Belgium). A P-value less than 0.05 was regarded as 3 77.2 20.4
4 85.5 29.0
statistically significant.
5 79.4 29.2
Mean 78.64 22.56
RESULTS SD 5.86 6.14
CV 7.45 27.22
Arachidonic acid
1. Precision 1 94.2 6.8
The results of precision testing are shown in Table 1. The CVs 2 91.9 2.4
using normal samples were 7.45% and 3.27% for ADP and arachi- 3 92.3 -0.4
4 87.7 0.4
donic acid, respectively. However, the CVs using patient samples
5 95.7 2.3
were 27.22% and 121.35% for ADP and arachidonic acid, respec- Mean 92.36 2.3
tively. SD 3.02 2.79
CV 3.27 121.35

Table 2. Stability test of CS-5100 platelet aggregation assay

Groups Agonist Sample No. 0 hr 1 hr 2 hr 4 hr 8 hr
Normal ADP 1 97.6 85.6 89.0 63.3 35.6
2 92.7 73.5 73.6 40.9 28.8
3 88.9 91.4 92.5 86.6 63.9
Mean 93.1 83.5 85.0* 63.6 42.8
Arachidonic acid 1 85.7 94.2 91.9 95.5 1.9
2 85.9 83.8 82.7 85.6 3.4
3 93.7 90.2 89.6 83.6 77.8
Mean 88.4 89.4 88.1 88.2* 27.7
Patient ADP 1 71.5 76.4 67.7 68.0 58.3
2 67.7 69.1 65.5 60.6 -23.0
3 42.0 48.4 47.2 39.0 26.3
Mean 60.4 64.6 60.1 55.9* 20.5
Arachidonic acid 1 1.4 0.5 2.3 2.4 1.7
2 2.3 1.7 1.2 1.6 -0.3
3 8.1 1.5 -0.5 1.6 1.8
Mean 3.9 1.2 1.0 1.9 1.1
*Stability was maintained for 2 hr or 4 hr in those samples.

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 안규대 외: Platelet Aggregation Test Using CS-5100

(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

ADP Arachidonic acid

100 100
CS-5100 aggregation (%)

CS-5100 aggregation (%)

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 (%)

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.

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안규대 외: Platelet Aggregation Test Using CS-5100

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

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 안규대 외: Platelet Aggregation Test Using CS-5100

요 약 ing of platelet function. Br J Haematol 2014;165:165-78.

3. Konkle BA. Acquired disorders of platelet function. Hematology Am
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기능장애를 진단하는 데 널리 사용되는 검사이다. CS-5100 (Sys- 4. Born GV and Cross MJ. The aggregation of blood platelets. J Physiol
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검사도 시행 가능하다. 저자들은 검사실에서의 CS-5100의 혈소판 5. Harrison P. Platelet function analysis. Blood Rev 2005;19:111-23.
응집검사의 성능을 평가하였다. 6. Cattaneo M, Hayward CP, Moffat KA, Pugliano MT, Liu Y, Michelson
방법: 저자들은 CS-5100의 정밀도, 안정성, 희석검사, 그리고 전통 AD. Results of a worldwide survey on the assessment of platelet func-
적인 장비와의 상관성을 평가하였다. 정밀도는 정상 및 환자 검체 tion by light transmission aggregometry: a report from the platelet phys-
로 시행하였다. 안정성은 8시간 동안 5번 측정하여 평가하였다. 희 iology subcommittee of the SSC of the ISTH. J Thromb Haemost 2009;
석검사는 ADP시약을 사용하여 정상 검체를 이용하여 평가하였다. 7:1029.
또한 전통적인 응집검사 장비와의 상관성을 평가하기 위해 Chrono- 7. Sakayori T, Watanabe Y, Nakajima K, Misawa E, Kobayashi K, Kurono
log사의 응집검사 장비(Chrono-Log Co., USA)와 5개의 시약을 각 H, et al. Introduction and evaluation of light transmission platelet ag-
각 10개의 검체를 이용하여 비교하였다. 저자들은 또한 22-30개의 gregation method on the Sysmex CS-series automated coagulation an-
정상 검체를 이용하여 5가지 시약의 참고범위도 측정하였다. alyzer. Sysmex Journal International 2016;26:1-10.
결과: 정상 검체에서의 변이계수는 ADP시약에서는 7.45%, Arachi- 8. Cattaneo M, Cerletti C, Harrison P, Hayward CP, Kenny D, Nugent D,
donic acid 시약에서는 3.27%였다. 안정성은 대부분의 검체에서 2 et al. Recommendations for the standardization of light transmission
시간까지 유지되었다. 희석검사 또한 1/2 희석배수까지 비슷한 결 aggregometry: a consensus of the working party from the platelet phys-
과를 보였다. Ristocetin시약을 제외한 나머지 시약에서는 모두 iology subcommittee of SSC/ISTH. J Thromb Haemost 2013;11:1183-9.
Chrono-log 장비와 강한 상관성을 나타내었다. CS-5100에서의 5가 9. Clinical and Laboratory Standard Institute. Evaluation of precision of
지 시약의 참고범위는 Chrono-log사의 값과 비슷하였다. quantitative measurement procedures; Approved guideline-third edi-
결론: 전통적인 장비와 비교하였을 때, CS-5100은 혈소판 응집검 tion. CLSI document EP05-A3. Wayne, PA: Clinical and Laboratory
사에서 신뢰할 만한 결과를 보였다. CS-5100은 응고검사와 혈소판 Standard Institute, 2014.
응집검사를 동시에 수행할 수 있는 장비이다. 그러므로, CS-5100 10. Clinical and Laboratory Standard Institute. Measurement procedure
의 사용은 검사비용을 줄이고 검사 소요시간을 단축시킬 수 있을 comparison and bias estimation using patient samples; Approved guide-
것이다. line-third edition. CLSI document EP09-A3. Wayne, PA: Clinical and
Laboratory Standard Institute, 2013.
Conflicts of Interest 11. Clinical and Laboratory Standard Institute. Defining, establishing,
and verifying reference intervals in the clinical laboratory; Approved
None declared. guideline-third edition. CLSI document EP28-A3C. Wayne, PA: Clini-
cal and Laboratory Standard Institute, 2010.
Acknowledgements 12. Taylor R. Interpretation of the correlation coefficient: a basic review. J
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This work was supported by the National Research Foundation of 13. Mukaka MM. Statistics corner: A guide to appropriate use of correla-
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