Influence of pre-analytical time and temperature conditions on serum thromboxane B2 levels
J.J.K. van Diemen¹, W.W. Fuijkschot¹, K. Spit¹, A.V.R van Reuler¹, T.N. Bonten², M.E. Numans², J.G.
van der Bom3,4, Y.M. Smulders¹ , A. Thijs¹
¹ Department of internal medicine, Amsterdam VU University Medical Center, Amsterdam, the Netherlands
² Department of Public Health & Primary Care, Leiden University Medical Center, Leiden, the Netherlands
3 Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, the Netherlands
4 JJ van Rood Center for Clinical Transfusion Research, Sanquin Research, Leiden, the Netherlands
Corresponding author Jeske (J.J.K.) van Diemen E: jj.vandiemen@vumc.nl T: 0031-630179557 Fax: 003120-4443349
Address: De Boelelaan 1118 (4A-45), 1081 HZ Amsterdam, the Netherlands
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1. Introduction
Aspirin is the oldest known platelet inhibitor and is able to reduce ischemic events by as much as 20- 25% (1). It does so by irreversibly blocking cyclo-oxygenase 1 (COX-1) and thereby preventing arachidonic acid from gaining access to the catalytic site of the enzyme (2). Without this access, thromboxane A2 cannot be formed and therefore the platelet misses its key promotor of aggregation (3). Despite aspirin therapy, there is a 3-9% yearly recurrence rate of ischemic events in
cardiovascular patients, which clinicians may refer to as ‘clinical aspirin resistance’ (3, 4). Furthermore, the concept of ‘biochemical aspirin resistance’ has been coined as inadequate platelet inhibition as determined by laboratory methods (5).
Although several different methods are available to assess ‘biochemical aspirin resistance’ (5, 6), serum thromboxane B2 (sTxB2) has been proposed as the reference test. In order to mimic natural thromboxane production incubation at 37 °C for 60 minutes is generally performed. However, the influence of pre-analytical time and temperature until incubation on sTxB2 levels is largely unknown (7- 9). This is of clinical importance, because differences in pre-analytical conditions could potentially explain divergent results of landmark cohort studies in this field. For example, the BOSTON-study showed a significant association of sTxB2 with major adverse cardiovascular events (17-19), whereas the ADRIE-study did not (10-12). Of note, sTXB2 levels differed between these studies as much as a 10-fold, which could not solely be explained by analytical variance or patient characteristics.
Unfortunately, pre-analytical sample conditions were not reported in either report. Therefore, the primary objective of our study was to assess the impact of pre-analytic time and temperature conditions on the level of sTxB2, in healthy subjects off and on aspirin therapy.
2. Materials and methods
We conducted an open label randomized cross-over study, comprising 12 healthy subjects who were enrolled in an ongoing study (registered in the Dutch trial register NTR5114). The study was
conducted in accordance with the Helsinki II Declaration. Written informed consent was obtained from all participants. The protocol was approved by the medical ethical committee of the VU University Medical Center Amsterdam. Participants were not eligible if their platelet count was <150 or > 400 × 109, if there was recent use of (other) antiplatelet drugs, anticoagulants or other drugs known to alter platelet function (e.g. NSAID’s, serotonin reuptake inhibitors).
All subjects were measured off aspirin therapy at time point zero, and again after 10 days of aspirin (acetylsalicylic acid, 80 mg, non-enteric-coated) intake at 8.00 PM. All measurements took place at 8.00 AM (i.e. 12 hours after aspirin intake) (figure 1.). Consumption of alcohol was prohibited within 24 hours prior to the measurement.
All venepunctures took place at the laboratory facility of the VU University Medical Center. Samples were drawn from the antecubital vein through a 21-gauge needle, first into several serum Clot Activator tubes (BD Vacutainer® Clot Activator Tube REF 368815) and last into one EDTA tube (BD Vacutainer® K2E (EDTA) 7.2mg REF 368861). The EDTA sample was used to measure several haemostatic values, namely platelet- , leucocyte- , thrombocyte count, haemoglobin level and percentage of reticulated platelets.
All five samples, off and on aspirin therapy, were handled accordingly to the following protocol (Figure 2.). Sample 1 was incubated for 60 minutes at 37 °C. Sample 2 was directly centrifuged after
venepuncture without incubation. Sample 3 was left at room temperature (circa 20°C) for 60 minutes.
Sample 4 was left at room temperature for 30 minutes and subsequently put in the 37°C incubator for 58
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the remaining 30 minutes. Finally, sample 5 was left at room temperature for 30 minutes and
subsequently put in the 37 °C incubator for the full remaining 60 minutes. After incubation all samples were immediately centrifuged at 3000 x g for 10 minutes and stored at -80 °C. After a maximum storage time of 12 months, during which sTxB2 levels have been reported to remain stable (7), samples were analyzed with Thromboxane B2 Express EIA Kit (distributed by Sanbio, Uden, the Netherlands, lot #0493751). All samples were analyzed in duplicate with a coefficient of variance below <15%. Furthermore, we used the same batch and one plate per subject. The assay had a range from 15.6-2,000 pg/ml and a sensitivity (80% B/B0) of approximately 45 pg/ml. When necessary, samples were diluted until a maximum of 12,000 pg/ml. Aspirin resistance was defined as >3,1 ng/ml residual sTxB2 level (8).
3. Statistical analysis
Continuous variables are expressed as mean ± standard deviation (SD) if normally distributed or median and interquartile range (IQR) for non-normally distributed variables. Data were compared by Friedman test for within group differences, by the Wilcoxon Signed Rank Test for non-normally distributed variables or by the paired T test for normally distributed variables. A p-value <0.05 was considered statistically significant. Depending on the distribution, missing values were replaced with either mean or median. Statistical analyses were performed with IBM SPSS statistics 22.0.
4. Results
4.1 Influence of time and temperature of incubation on sTXB2 off aspirin Participant characteristics are reported in table 1.
As shown in figure 2A and table 2, incubation at 37°C was necessary to maximize sTxB2 levels (12.00 ng/ml; IQR: 10.50-12.00). Keeping the sample at room temperature for 30 minutes prior to incubation did not affect sTxB2 levels compared with the reference condition (12.00 ng/ml; IQR:10.00-12.00), nor did shortening the incubation period from 60 to 30 minutes (12.00 ng/ml; IQR: 10.50-12.00).
4.2 Influence of time and temperature of incubation on sTxB2 on aspirin
Aspirin intake changed the effect of pre-analytical conditions on sTxB2 levels: sTxB2 was the highest after incubation for 60 minutes at 37°C (2.46 ng/ml; IQR: 2.08 -3.17), but was markedly lower after keeping the sample at room temperature for 60 minutes (0.07 ng/ml; IQR: 0.04-0.10), or for 30
minutes at room temperature followed by 60 minutes at 37 °C (0.60 ng/ml; IQR: 0.32-1.12). As well as after a shorter (30 at room temperature and 30 at 37 °C ) incubation period (0.26 ng/ml; IQR: 0.20- 0.89). Furthermore, there was a marked difference in the amount of samples characterized as aspirin resistant when compared with the reference method (33.3%): no incubation (0%); 60 min. at room temp. (8.3%); 30 min. at room temp. and 30 min. at 37°C (8.3%); 30 min. at room temp. and 60 min. at 37°C (0%). Details are depicted in table 3 and figure 3B & 4.
5. Discussion
Our study clearly demonstrates that the level of sTxB2 can be markedly decreased by changing pre- analytic time and temperature conditions. Moreover this decrease seems more apparent on aspirin therapy.
Petrucci et al. have previously shown a significant influence of delay until incubation (8). Nevertheless, blood is often not drawn next to the incubators. Thus there might be a biased view of aspirin efficacy in past research. For example, the 10 fold difference of the median sTxB2 levels between the BOSTON- study and ADRIE-study (0,6 and 7 ng/ml) might be explained by a larger time until incubation (10-12).
Surprisingly, it is not common practice to report ‘time until incubation’.
In our opinion sample handling condition #5 is best comparable to the everyday practice: there is a delay until a sample reaches the 37 °C incubator and subsequently the sample is incubated for the full standard of 60 minutes. As can be seen in figure 3B, the on aspirin samples have statistically
significantly decreased sTxB2 levels compared with 60 min. incubation at 37 °C. This is further
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illustrated by the fluctuation in aspirin ‘resistant’ samples (fig. 4).This finding is of clinical importance because patients who have been labeled "aspirin resistant" have a significantly higher rate of myocardial infarction (MI) and/or stroke(13). Although personalized antiplatelet therapy based on platelet testing is not yet common practice, mislabeling of aspirin resistance could be of serious clinical consequence in the near future. We hypothesize that the decrease in sTxB2 levels is created by the passing of the ‘aggregation window’. Uninhibited thrombocytes are able to reach full activation through several activation pathways (e.g. adenosine diphosphate, fibrinogen, thrombin, epinephrine, collagen receptor pathway)(14), facilitating rapid and full aggregation even under suboptimal circumstances such as ex-vivo. However, thrombocytes inhibited by aspirin may require more time to reach full aggregation under similar circumstances.
In this article, we therefore propose to standardize the time and temperature until incubation. For example by incorporating a standard delay of 10 minutes at room temperature. This would limit the intra-research group variables and thus, if reported in future publications, increase the inter-research group comparability.
When viewing the results of this study one can wonder whether it is necessary to perform full
incubation (i.e. 60 min. incubation at 37 °C) to measure the effect of aspirin on thrombocytes (9). For example, the samples without incubation off and on aspirin still differed by a mere 10-fold. Moreover, the samples incubated at room temperature differed by a 62-fold. Thus, even under suboptimal circumstances it is possible to detect a difference in certain regimens. Therefore, by enforcing the full 60 min. incubation at 37 °C there is too much variation caused by a difference in delay until
incubation. Merely incubating at room temperature could be a solution. However, those sTxB2 levels are of course not comparable to results in other studies generated under different incubation temperatures.
In conclusion, different sample handling conditions can markedly decrease the level of sTxB2 . This decrease seems more apparent on aspirin therapy, emphasizing the importance of strict sample handling protocols, particularly in studies on aspirin effects.
6. Funding
This research did not receive any specific grant from funding agencies in the public, commercial, nor for not-for-profit sectors.
7. Acknowledgment
First, we would like to express our gratitude towards all individuals who participated in the study.
Furthermore, we thank the laboratory technicians Annelies Hoenderdos and Petra Noordijk, and Hilde Hopman and Wilma Rosendal, of the hemostasis laboratory of the Leiden University Medical Center &
the VU University Medical Center, respectively.
8. Conflict of interest
No conflict of interest identified.
9. References
1. Patrono C. Low-dose aspirin in primary prevention: cardioprotection, chemoprevention, both, or neither? Eur Heart J. 2013;34(44):3403-11.
2. Floyd CN, Ferro A. Mechanisms of aspirin resistance. Pharmacol Ther. 2014;141(1):69-78.
3. Antithrombotic Trialists C. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ.
2002;324(7329):71-86.
4. Antithrombotic Trialists C, Baigent C, Blackwell L, Collins R, Emberson J, Godwin J, et al.
Aspirin in the primary and secondary prevention of vascular disease: collaborative meta-analysis of individual participant data from randomised trials. Lancet. 2009;373(9678):1849-60.
5. Michelson AD. Aspirin resistance. Pathophysiol Haemost Thromb. 2006;35(1-2):5-9.
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6. Michelson AD, Cattaneo M, Eikelboom JW, Gurbel P, Kottke-Marchant K, Kunicki TJ, et al.
Aspirin resistance: position paper of the Working Group on Aspirin Resistance. J Thromb Haemost.
2005;3(6):1309-11.
7. Sadilkova L, Paluch Z, Mottlova J, Bednar F, Alusik S. The effect of selected pre-analytical phase variables on plasma thromboxane A(2) measurements in humans. Int J Lab Hematol.
2013;35(1):92-100.
8. Petrucci G, Rizzi A, Cavalca V, Habib A, Pitocco D, Veglia F, et al. Patient-independent variables affecting the assessment of aspirin responsiveness by serum thromboxane measurement.
Thromb Haemost. 2016;116(5):891-6.
9. Patrono C, Ciabattoni G, Pugliese F, Pinca E, Castrucci G, De Salvo A, et al.
Radioimmunoassay of serum thromboxane B2: a simple method of assessing pharmacologic effects on platelet function. Adv Prostaglandin Thromboxane Res. 1980;6:187-91.
10. Frelinger AL, 3rd, Li Y, Linden MD, Barnard MR, Fox ML, Christie DJ, et al. Association of cyclooxygenase-1-dependent and -independent platelet function assays with adverse clinical outcomes in aspirin-treated patients presenting for cardiac catheterization. Circulation.
2009;120(25):2586-96.
11. Fontana P, Berdague P, Castelli C, Nolli S, Barazer I, Fabbro-Peray P, et al. Clinical
predictors of dual aspirin and clopidogrel poor responsiveness in stable cardiovascular patients from the ADRIE study. J Thromb Haemost. 2010;8(12):2614-23.
12. Reny JL, Berdague P, Poncet A, Barazer I, Nolli S, Fabbro-Peray P, et al. Antiplatelet drug response status does not predict recurrent ischemic events in stable cardiovascular patients: results of the Antiplatelet Drug Resistances and Ischemic Events study. Circulation. 2012;125(25):3201-10.
13. Gum PA, Kottke-Marchant K, Welsh PA, White J, Topol EJ. A prospective, blinded
determination of the natural history of aspirin resistance among stable patients with cardiovascular disease. J Am Coll Cardiol. 2003;41(6):961-5.
14. Jennings LK. Mechanisms of platelet activation: need for new strategies to protect against platelet-mediated atherothrombosis. Thromb Haemost. 2009;102(2):248-57.
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Figure 1. Flowchart of protocol
Figure 2. Flowchart of sample handling
Figure 3. sTxB2 levels in ng/ml
A. Sample variations of off aspirin samples; B. Sample variations of on aspirin samples 288
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† Data are presented as median and interquartile range (IQR)
Figure 4. Percentages categorized as aspirin ‘resistant’ per sample variation
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Table 1. Baseline characteristics of study sample
Healthy subjects (n=12) General characteristics
Female sex (n, %) 7 (58.3)
Age (years) 22.3 ± 1.6
Caucasian (n,%) 11 (91,7)
BMI (kg/m2) 21.3 ± 1.4
Current smoker (n, %) 1 (8.3)
Alcohol intake (units/week) 1.2 ± 0.4
Coffee intake (units/day) 2.3 ± 1.6
Systolic blood pressure (mmHg) 118.3 ± 4.6
Diastolic blood pressure (mmHg) 72.4 ± 2.0
Reported time of intake of aspirin 19:52 ± 45 min.
Medication usage in past month (n,%)
NSAIDS 0 (0)
Antibiotics 0 (0)
SSRI 0 (0)
Hormonal anticonception 5 (58.3)
Laboratory values
Hemoglobin level (mmol/l) 8.5 ± 0.8
Thrombocyte count (109/L) 255.6 ± 61.8
Leukocyte count (109/L) 7.6 ± 2.2
RTP in percentages 1.5 ± 0.7
1 Non-normally distributed parameters are presented as median and interquartile range (IQR).
2 Normally distributed parameters are presented as mean and standard deviation (±).
BMI; body mass index, NSAIDs; non-steroidal anti-inflammatory drug, SSRI; selective serotonin reuptake inhibitor, RTP; reticulated platelets.
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Table 2. Overview of sTxB2 levels off aspirin
Incubation temperature and time sTxB2 level sTxB2 level after 60 minutes at 37 °C
No incubation 0.56 (0.37-1.02)² 12.00 (10.50-12.00) p=0.002
*
Room temperature for 60 minutes 4.39 (3.77-7.24) “” p=0.003
Room temperature for 30 minutes and 30 minutes at 37 °C
12.00 (10.00-12.00)
“” p=0.180
Room temperature for 30 minutes and 60
minutes at 37 °C 12.00
(10.50-12.00) “” p=0.655
* The Wilcoxon Signed Rank Test was used to test for in between group differences; 1 Missing values were replaced with median; ² Variables are expressed in ng/ml; 3 Data are presented as median and interquartile range (IQR)
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Table 3. Overview of sTxB2 levels on aspirin
Incubation temperature and time sTxB2 level sTXB2 level after 60
minutes at 37 °C P value
No incubation 0.05 (0.03-
0.11)2 2.46 (2.08 -3.17) 0.003*
Room temperature for 60 minutes 0.07 (0.04-
0.10) “” 0.005
Room temperature for 30 minutes and 30
minutes at 37 °C 0.26 (0.20-
0.89) “” 0.003
Room temperature for 30 minutes and 60 minutes at 37 °C
0.60 (0.32- 1.12)
“” 0.003
* The Wilcoxon Signed Rank Test was used to test for in between group differences; 1 Missing values were replaced with median; ² Variables are expressed in ng/ml; 3 Data are presented as median and interquartile range (IQR)
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