http://informahealthcare.com/plt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, 2015; 26(2): 132–137 ! 2015 Informa UK Ltd. DOI: 10.3109/09537104.2014.898141
In vitro assessment of platelet concentrates with multiple electrode aggregometry Caroline Shams Hakimi1*, Camilla Hesse2,3*, Ha˚kan Walle´n4, Fredrik Boulund5, Ammi Grahn2, & Anders Jeppsson1,6 1
Department of Cardiothoracic Surgery, Sahlgrenska University Hospital, Gothenburg, Sweden, 2Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, 3Regional Blood Bank, Sahlgrenska University Hospital, Gothenburg, Sweden, 4Karolinska Institute, Department of Clinical Sciences, Danderyd Hospital, Stockholm, Sweden, 5 Department of Mathematical Sciences, Division of Mathematical Statistics, Chalmers University of Technology and University of Gothenburg, Gothenburg, Sweden, and 6Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden ABSTRACT
Storage impairs platelet function. It was hypothesized that multiple electrode aggregometry in vitro could be used to follow aggregability in platelet concentrates over time and that the results predict the efficacy of platelet transfusion in an ex vivo transfusion model. In vitro platelet aggregability was assessed in apheresis and pooled buffy coat platelet concentrates (BCs) (n ¼ 13 each) using multiple electrode aggregometry with different agonists 1, 3, 5 and 7 days after preparation. In the ex vivo transfusion model, whole blood samples from nine healthy volunteers were collected every second day. The samples were supplemented with stored platelets (+146 109 l1) from the same unit 1, 3, 5 and 7 days after preparation. Platelet aggregability was assessed in the concentrate and in the whole blood samples before and after platelet supplementation. There was a continuous reduction in in vitro platelet aggregability over time in both apheresis and pooled BCs. The same pattern was observed after ex vivo addition of apheresis and pooled BCs to whole blood samples. The best correlation between in vitro aggregability and changes in aggregation after addition was achieved with collagen as agonist (r ¼ 0.67, p50.001). In conclusion, multiple electrode aggregometry can be used to follow aggregability in platelet concentrates in vitro, and the results predict with moderate accuracy changes in aggregation after addition of platelet concentrate to whole blood samples.
Aggregometry, apheresis, buffy coat, platelets
Introduction Platelet transfusion is used to increase the number of functional platelets in patients with hereditary or acquired thrombocytopenia and in patients with impaired platelet function due to antiplatelet therapy or disease, or to improve hemostasis in patients with ongoing bleeding. After preparation, the platelet concentrate is stored until use. However, the function of the stored platelets deteriorates rapidly with time. Within days, there are marked functional and morphological changes in the concentrate. This condition is generally referred to as platelet storage lesion [1–3]. In vitro monitoring of platelet function may improve the use of platelet concentrates, but currently there is no generally accepted method. A number of different in vitro methods have been tried but none have gained widespread clinical use [4–7]. It has also been difficult to relate the results of in vitro testing to the clinical efficacy after transfusion [8,9]. One possibility for assessment of
*These two authors contributed equally to the study Correspondence: Dr. Anders Jeppsson, Department of Cardiothoracic Surgery, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden. Tel: +46 31 3427515. Fax: +46 31 41799. E-mail: [email protected]
History Received 12 November 2013 Revised 20 January 2014 Accepted 20 February 2014 Published online 4 July 2014
platelet function in vitro is to measure the aggregation of the platelets in response to specific agonists. Light transmission aggregometry, the gold standard of clinical platelet function testing, uses this principle in platelet-rich plasma, and some novel point-of-care devices also use the same principle in whole blood samples. One of these new techniques is multiple electrode aggregometry (MEA; MultiplateÕ , Verum Diagnostica GmbH, Munich, Germany) [10,11]. With this method, aggregation is induced in the sample with an agonist, e.g. collagen or adenosine diphosphate (ADP). The aggregation increases the impedance between two electrodes in a test cell. The results are displayed as an aggregation curve where the area under the curve is a measure of aggregation. Until now, MEA has mainly been used to test platelet inhibition in patients on antiplatelet therapy [11,12] but it has also been tested in a few studies to directly assess in vitro aggregation of stored platelets. Jilma-Stohlawetz et al. used the method to compare different apheresis devices and to study the effect of plasma removal on platelet aggregation [13,14] and Ostrowski et al. evaluated the effect of pathogen reduction technologies . The latter study also found deterioration in platelet aggregation over time in platelet concentrates, as measured with MEA, but no previous studies have attempted to examine the relationship between in vitro aggregation and platelet function in an ex vivo transfusion model.
Aggregometry in stored platelets
We hypothesized that MEA can be used to assess platelet concentrate aggregation both in vitro and in a transfusion model, and that the results of the in vitro analysis would predict the results in the transfusion model. To test this hypothesis, two prospective descriptive studies were designed, which also evaluated the effect of storage time.
Methods Study subjects Study A. In vitro platelet aggregation in apheresis platelet concentrates and buffy coat platelet concentrates Platelets prepared with two different techniques (see below) and stored for 1, 3, 5 or 7 days were investigated (n ¼ 13 for each). All platelets were derived from blood donors at the regional blood bank, Sahlgrenska University Hospital. The blood donors were selected according to the standard procedures of the blood bank. The donors gave written informed consent for the use of the blood components both in study A and B. Study B. Platelet aggregation in whole blood after addition of apheresis platelet concentrate or BC Nine healthy men (mean age 40 ± 3.6 years) were included in the study after giving informed written consent. To prevent reactions due to alloantibodies when platelet concentrate was added, only men without any previous history of transfusion were included. Subjects were instructed to avoid ingestion of drugs influencing platelet function (i.e. aspirin and NSAIDs) 1 week before blood sampling. The study was approved by the Regional Research Ethics Committee in Gothenburg, Sweden, and was performed according to the ethical principles of the Declaration of Helsinki.
according to the manufacturer’s instruction. Prior to the donation the platelet count of the donor was at least 230 109 l1. The target concentration was 1600 109 l1 for each donation. The whole blood to citrate ratio was 10:1 and the platelets were stored in autologous plasma throughout the storage time. The levels of A- and B-antibodies were checked in the concentrate of blood group O; only concentrates with a low level of antibodies (titer of51/100 with indirect antiglobulin testing) were included in the ex vivo study. Study protocol Study A. In vitro platelet aggregation in ACs and BCs Samples from both types of concentrates were taken aseptically from the bag using a separate sampling bag on day 1, 3, 5 and 7 after collection. The samples were transferred to EDTA tubes (Vacuette K2E K2EDTA, 3 ml; Greiner Bio-One GmbH, Kremsmu¨nster, Austria) for measurement of platelet count and mean platelet volume (MPV), and to small plastic tubes without any additives for assessment of platelet aggregation. Platelet count and MPV was analyzed within 1 hour after sampling. A visible ‘‘platelet swirl’’ was detected in each platelet concentrate before every sample collection. The concentrates (n ¼ 13 for each collection day and platelet type) were analyzed with MEA. For ACs, 150 ll of the concentrate together with 150 ll phosphate buffered saline (PBS; 140 mM NaCl, 10 mM Na3PO4, pH 7.4) was used in the assay. For pooled buffy coat concentrates, PBS was replaced with 150 ll allogeneic plasma of blood group AB. Study B. Platelet aggregation in whole blood after addition of AC or BC
The buffy coat-derived platelet concentrates were prepared from buffy coats donated by four regular donors of whole blood on the day before preparation. Whole blood units were collected in bottom-and-top bags (MacoPharma Nordic AB, Helsingborg, Sweden) containing 63 ml citrate phosphate dextrose as anticoagulant. The whole blood units were hard-spin-centrifuged (4880 g for 11 minutes at 23 C) and the buffy coat was separated from the red blood cells and the plasma using a blood expander platform (Macopress Smart, MacoPharma Nordic AB). Next, the buffy coats were pooled together with platelet additive solution (SSP; MacoPharma Nordic AB) and the pooled material was processed in an automated blood component processing device (TACSI, Terumo BCT Europe, Zaventem, Belgium) to separate the remaining red cells. The residual plasma content was 20%.
Whole blood samples were collected from nine healthy subjects every second day: from the antecubital vein using a BD Vacutainer Eclipse blood collection needle (BD Diagnostics, NJ). The blood samples were collected in 3 ml hirudin tubes (0.15 mg l1; Verum Diagnostica GmbH, Munich, Germany). Before analysis, the samples were diluted (20% by volume) with hydroxyethyl starch (HES) (Venofundin 60 mg/ml; B. Braun Melsungen AG, Melsungen, Germany), to mimic samples from patients with impaired platelet function. ACs and BCs were prepared as reported above. Samples for MEA were collected from the concentrates 1, 3, 5 and 7 days after the platelets were donated. Each concentrate was used to supplement whole blood samples from three study subjects. The whole blood samples always received platelet concentrates from the same preparation. For each study subject and day, three samples were prepared for platelet aggregation tests: one baseline sample, one sample supplemented with AC and one supplemented with BC. The baseline sample consisted of 1080 ll HES-diluted blood and 285 ll PBS (to control for the diluting effect of platelet concentrate), while the platelet-supplemented samples consisted of 1080 ll of HES-diluted blood and 285 ll platelet concentrate. The amount of platelets in the concentrates was standardized to 700 109 l1. If the true platelet concentration was higher, the platelet concentrate was diluted with PBS to give the desired concentration. The amount of platelets added was 2 108, corresponding to an increase of 146 109 l1. A sample for hemoglobin, hematocrit and platelet count analysis was also collected from the healthy individuals in an EDTA tube.
Apheresis platelet concentrates
Apheresis platelet concentrates were obtained by a standard apheresis procedure (Trima Accel; Terumo BCT Europe)
Platelet aggregation was measured with MEA as previously described [10,17]. For each of the three samples, the tests ADP,
Platelet concentrates Two types of platelet concentrate were used: buffy coat-derived (BC) and apheresis platelet concentrates (ACs). The concentrates were processed by the regional blood bank and were of blood group O or A for study A, and group O for study B. All platelet concentrates were leucocyte-reduced (51 106 leucocytes per unit) and stored at 22 C in a platelet incubator (Helmer Agitator; Fenwal Europe, Mont Saint Guibert, Belgium) with horizontal agitation until use. All preparations were conducted in accordance with the current European guidelines . Buffy coat platelet concentrates
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ASPI, TRAP and COL were used. The ADP test assesses the P2Y12 receptor-dependent aggregation with ADP (6.5 mM) as agonist and the ASPI test assesses cyclooxygenase-dependent aggregation with arachidonic acid (AA) (0.48 mM) as agonist. The TRAP test assesses thrombin receptor- (PAR-1) dependent platelet aggregation with thrombin receptor-activating peptide (TRAP)-6 (32 mM) as agonist. The COL test assesses cyclooxygenase-dependent aggregation with collagen (3.2 mg l1) as agonist. The area under the aggregation curve (AU minute, 6minute measurement) was taken as a measure of platelet aggregation. Hemoglobin, hematocrit and platelet count were analyzed using clinical standard methods. Statistics All data are presented as mean ± SEM. Any p value of50.05 was considered statistically significant. Estimation of the reduction in aggregation over time was done using linear regression based on the aggregation data. For the ex vivo study, regression analysis was performed on data after adjusting for the temporal dependency of the added platelet concentrate by subtraction of day 1 values from all samples. The correlation between in vitro aggregation and changes in whole blood aggregation after supplementation with platelet concentrate was examined with Spearman rank-sum test on pooled data from all four time points. Variables at different time points were compared with Student’s t-test or paired t-test when appropriate. Statistical analyses were performed with R software (version 2.13.1) and IBM SPSS version 19 (SPSS Inc.).
Results Study A. In vitro platelet aggregation in ACs and BCs In buffy coat preparations, there was a significant reduction in platelet aggregation over time with all four agonists (all p50.001) (Figure 1 and Table SI). At day 7, ADP- and AA-induced aggregation was 5% and 12%, respectively, of the aggregation at day 1. Collagen- and TRAP-induced aggregation at day 7 was 21% and 65%, respectively, of the aggregation at day 1. Figure 1. ADP-, AA-, TRAP- and COL-induced platelet aggregation in buffy coat platelet concentrates and apheresis platelet concentrates after 1, 3, 5 and 7 days storage. Buffy coat concentrates (BC) are depicted with solid line and filled circles (n ¼ 13 at each time point). Apheresis concentrates (AC) are depicted with dashed line and open circles (n ¼ 13 at each time point). Shown are mean values and SE of the raw aggregation measures (AUC, area under curve), along with a linear regression model fitted to the data.
Platelets, 2015; 26(2): 132–137
In apheresis preparations, there was a significant reduction over time in ADP- (p50.001), AA- (p ¼ 0.005) and collagen(p50.001) induced aggregation, while TRAP-induced aggregation did not change significantly (p ¼ 0.20) (Figure 1 and Table SI). At day 7, ADP-, AA- and collagen-induced aggregation were 14%, 80% and 22%, respectively, of the aggregation at day 1. Study B. Platelet aggregation in whole blood after addition of AC or BC The three platelet concentrates used in the ex vivo transfusion model showed the same pattern as in the in vitro study, with continuous reductions over time in aggregation with all agonists in the buffy coat preparations and reductions in ADP-induced aggregation in the apheresis preparations. Hemoglobin concentration, hematocrit and platelet counts in the nine study subjects (147 ± 2.3 g l1, 0.43 ± 0.01 l l1 and 222±21 109 l1, respectively, at day 1) and baseline platelet aggregation (Table I) were stable over the four sampling time points. Addition of platelets from buffy coat preparations improved AA- and TRAP-induced aggregation in the whole blood samples at all four time points, but the effects deteriorated with storage time (Table I, Figure 2 and Figure S1). Addition of buffy coat platelets improved COL-induced aggregation at day 1 and day 3 but did not influence aggregation on day 5 and 7. ADP-induced aggregation improved at day 1 but was reduced after supplementation with buffy coat platelets after 3, 5 and 7 days. Also, the changes in COL- and ADP-induced aggregation deteriorated with storage time. Addition of platelets from apheresis preparations improved AA-, TRAP- and COL-induced aggregation in the whole blood samples at all four time points, without any significant deterioration over time (Table I, Figure 2 and Figure S1). ADP-induced aggregation improved after platelet supplementation at day 1, was unchanged at day 3 and was reduced after 5 and 7 days. Using pooled data from all four time points, there were significant correlations between in vitro aggregability and changes in aggregation after ex vivo transfusion of platelets from buffy coat preparations for ADP-induced (r ¼ 0.61,
Aggregometry in stored platelets
p50.001), AA-induced (r ¼ 0.66, p50.001) and COL-induced (r ¼ 0.77, p50.001) aggregation while there was no correlation with TRAP-induced aggregation (Table SII). For apheresis preparations, ADP- (r ¼ 0.36, p ¼ 0.036) and COL-induced (r ¼ 0.49, p ¼ 0.003) aggregation correlated significantly but not AA- and TRAP-induced aggregation. When apheresis and buffy coat results were combined, the following correlation coefficients Table I. Platelet aggregation in whole blood samples from healthy volunteers before dilution, at baseline after dilution with hydroxyethyl starch and after addition of buffy coat platelet concentrate (n ¼ 9) or apheresis platelet concentrate (n ¼ 9) stored for 1, 3, 5 or 7 days, respectively. Mean ± SEM.
ADP (AU minute) Undiluted Baseline BC AC AA (AU minute) Undiluted Baseline BC AC TRAP (AU minute) Undiluted Baseline BC AC COL (AU minute) Undiluted Baseline BC AC
72 ± 4.9 45 ± 3.0*** 50 ± 5.5 53 ± 4.2
– 45 ± 3.2 37 ± 4.3 47 ± 2.8
– 42 ± 4.6 27 ± 3.9 41 ± 5.6
– 45 ± 4.5 27 ± 3.6 40 ± 3.2
82 ± 3.5 62 ± 3.4*** 92 ± 3.7 87 ± 2.1
– 60 ± 4.3 85 ± 4.7 87 ± 2.2
– 53 ± 5.4 68 ± 7.1 77 ± 8.1
– 61 ± 5.3 74 ± 4.8 85 ± 3.2
99 ± 5.4 81 ± 3.5*** 92 ± 3.9 93 ± 2.1
– 80 ± 5.1 89 ± 3.7 93 ± 2.7
– 75 ± 6.0 82 ± 3.4 89 ± 3.9
– 77 ± 5.6 82 ± 3.2 92 ± 3.9
84 ± 5.0 61 ± 3.4*** 77 ± 3.3 78 ± 3.2
– 60 ± 4.5 73 ± 4.1 75 ± 2.7
– 55 ± 5.1 53 ± 6.0 64 ± 5.3
– 56 ± 4.6 56 ± 3.2 68 ± 3.2
AA, arachidonic acid; AC, apheresis platelet concentrates; ADP, adenosine diphosphate; BC, buffy coat platelet concentrates; COL, collagen; SEM, standard error of the mean; TRAP, thrombin receptor activating peptide-6. ***p50.001 compared to undiluted sample.
Figure 2. ADP-, AA-, TRAP- and COL-induced platelet aggregation in whole blood samples from healthy volunteers supplemented with buffy coat platelet concentrates or apheresis platelet concentrates. Buffy coat concentrates (BC) are depicted with solid line and filled circles (n ¼ 9 at each time point). Apheresis concentrates (AC) are depicted with dashed line and open circles (n ¼ 9 at each time point). The baseline is aggregation in whole blood before addition of platelets. Note that lines shown are not regression lines. For the regression analysis of this data, see Supplementary Figure.
and p values were obtained; ADP: r ¼ 0.66, p50.001; AA: r ¼ 0.31, p ¼ 0.010; TRAP: r ¼ 0.28, p ¼ 0.019; COL: r ¼ 0.67, p50.001.
Discussion The main finding of the present study was that multiple electrode aggregometry can be used to follow platelet aggregability in vitro, and the results moderately well predict changes in aggregation in whole blood after platelet supplementation in an ex vivo platelet transfusion model. Platelet storage lesion and bacterial contamination limits the storage time for platelet concentrates to 5–7 days [3,18]. A number of different methods have been considered for in vitro quantification and characterization of platelet storage lesion [4–7]. These methods include both routine assays in transfusion practice and tests mainly used for research purposes. Generally, morphological and functional biomarkers show a consistent decline over time. However, so far no in vitro assay has gained widespread clinical use, probably because there is no strong evidence that in vitro data correlate with platelet recovery after transfusion in vivo [3,5,18]. This is further complicated by the fact that the destiny of transfused platelets will be dependent on the clinical state of the recipient, e.g. if it is a patient with ongoing bleeding or a patient receiving prophylactic treatment due to a hematological disease. In the present study, we hypothesized that in vitro platelet aggregability measured with MEA could be used to follow platelet function over time in platelet concentrates, and that the result would predict the changes in aggregation in an ex vivo transfusion model. In accordance with Ostrowski et al. , we found that in vitro platelet aggregability declined with time. Furthermore, we found that the in vitro results predicted with moderate accuracy the changes in aggregation in an ex vivo transfusion model with artificially impaired platelet aggregation. The best correlation was achieved with collagen as agonist. These results therefore suggest that MEA may be a useful tool to monitor platelet function in vitro. However, our results should be
C. Shams Hakimi et al.
reproduced with other markers of platelet function before the true value of in vitro platelet aggregometry can be established. Even though the in vitro study showed a decline in aggregation over time, addition of platelets to whole blood samples improved aggregation with most of the agonists also at the end of the storage period (Figure 2). This indicates that there is substantial recovery of platelets after transfusion, which might explain why it is difficult to show a correlation between in vitro and ex vivo data. There was, however, one potentially important exception. ADP-induced aggregation after ex vivo transfusion was in fact impaired after addition of buffy coat platelet concentrates already 3 days after preparation, and apheresis-prepared platelets had no effect from day 3 onward (Figure 2). This may in part be due to platelet degranulation, a phenomenon known to occur during platelet storage, and which is associated with release of ADP causing down-regulation of ADP-induced platelet responses . Notably, an increasing number of patients are treated with dual antiplatelet therapy using acetylsalicylic acid and an ADP-dependent platelet inhibitor (e.g. clopidogrel or ticagrelor). There is a well-known increased risk of surgical or non-surgical bleeding in these patients, and if a patient suffers a major bleeding, the first-line treatment will be platelet transfusion. We recently reported that the effect of platelet transfusion on ADP-dependent platelet aggregation ex vivo is limited in acute coronary syndrome patients on dual antiplatelet therapy with acetylsalicylic acid and clopidogrel or ticagrelor . One may thus speculate, based on the results of the present study that this patient category would particularly benefit from transfusion with fresh platelets. Interestingly, TRAP-induced and AA-induced platelet aggregation was remarkably well preserved also after 7 days storage in AC. Thus, two platelet activating pathways can still be functional in vivo and perhaps partly compensate for impaired ADP-dependent platelet responsiveness. The study has important limitations, but it also has strengths. Platelet aggregation in vitro and ex vivo was only assessed with one method, and it is possible that other methods would have yielded different results. Platelet count in the in vitro study differed significantly at day 5 compared to day 1 in AC preparations (Table S1). It is possible that platelet count may influence aggregometry results although this previously only has been shown with markedly lower counts (5100 109 l1) than in the present study . When we calculated the correlation between platelet count and aggregometry results in our material there was only a weak correlation between BC platelet count and TRAP-induced aggregation (r ¼ 0.29, p ¼ 0.035) while the all other aggregation tests in BC and AC preparations did not correlate significantly. The whole blood samples from the healthy study subjects were diluted with 20% HES to impair platelet function. This was done to mimic a clinical situation – bleeding in a patient with impaired platelet function. This method is widely used in hemostasis research [22,23], but we acknowledge that this is an artificial situation and that the results may be applicable only to this particular situation. Furthermore, the ex vivo platelet transfusion model lacks important factors compared to in vivo transfusion, such as the contribution of the vascular endothelium and blood flow to primary hemostasis. On the other hand, the model is reproducible, contains all the elements of blood, and study conditions can be standardized to a higher degree than during in vivo transfusion. One particular strength was the study design in the ex vivo transfusion model. Fresh whole blood samples from the same study subject were supplemented with platelets from the same unit on four occasions (1, 3, 5 and 7 days after donation) Thus, we have controlled both for differences between study subjects and differences between platelet concentrate units, which might otherwise have obscured the results.
Platelets, 2015; 26(2): 132–137
In conclusion, our results indicate that MEA performed directly on the platelet concentrate might be a useful tool for assessment of platelet aggregability in vitro and that the results with moderate accuracy predict aggregation in an ex vivo transfusion model. Further studies with other markers of platelet recovery after transfusion are warranted to validate our observations.
Acknowledgements The skillful assistance of research assistants Linda Thimour-Bergstro¨m, Christine Roman-Emanuel and Erika Backlund is gratefully acknowledged.
Authorship contributions C.S.H., C.H. and A.J. designed the study. C.H. and A.G. prepared all platelet concentrates. C.S.H. and C.H. performed all measurements, F.B. performed all statistical analyses. The data were analyzed by C.S.H., C.H., H.W. and A.J. C.S.H. and C.H. wrote the first draft of the manuscript. All the authors contributed with intellectual and scientific input to the final manuscript.
Declaration of interest The authors report no declarations of interest. The study was supported by the Swedish Heart and Lung Foundation (grant number 20090488 to A.J.) and Sahlgrenska University Hospital (ALF/LUA grant number 146281 to A.J.). The study sponsors had no influence on the analysis and interpretation of data, in the writing of the report, or in the decision to submit the paper for publication.
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Supplementary material available online Supplementary Table I Supplementary Table II Figure S1. Platelet concentrates added to whole blood Supplementary material can be viewed and downloaded at http://informahealthcare.com/plt
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