This work was done in The Laboratory of Internal Medicine II, School of Veterinary Medicine, Azabu University.
Previously presented at the 150th Meeting of the Japanese Society of Veterinary Science, 2010.
Corresponding author: Ryo Tsuchiya, DVM, PhD, The Laboratory of Internal Medicine II, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuou-ku, Sagamihara-shi, Kanagawa 252-5201, Japan; e-mail:email@example.com.
Platelet concentrates (PC) are prepared by centrifugation of platelet-rich plasma (PRP) that is prepared by centrifugation of whole blood. The resuspension of the platelet pellet during PC preparation from dogs is difficult because of platelet activation induced by centrifugation.
To investigate the efficacy of adding prostaglandin E1 (PGE1) to prevent platelet activation during PC preparation from dogs.
Fifteen healthy Beagle dogs.
Prospective, experimental trial: PGE1 was added to PRP before the high-speed centrifugation during PC preparation. To estimate the effect of this addition, we assessed the platelet aggregability before transfusion, the survival of the platelets after transfusion, and the platelet reactivity after transfusion, which is estimated by the P-selectin expression of the platelets when stimulated by thrombin.
The difficulty associated with platelet resuspension was resolved by PGE1.PGE1 strongly inhibited platelet aggregation induced by collagen and ADP; however, it recovered after the platelets were resuspended in plasma without PGE1 (mean aggregation ratio; collagen: 10.00–80.80%, ADP: 8.20–53.60%). Survival of the platelets after transfusion was not affected by PGE1 (mean 8.04 and 7.56 days, without and with PGE1), and thrombin-induced P-selectin expression after transfusion in PGE1-treated PC was also well maintained (mean positive ratio 53.7 and 47.9%, before and 24 hours after transfusion).
Conclusions and Clinical Importance
The addition of PGE1 in PRP before the centrifugation of PRP can improve the preparation efficiency of PC from dogs, while maintaining the therapeutic efficacy of the platelets.
Platelet transfusions are indicated when animals have bleeding tendencies associated with thrombocytopenia, thrombocytopathy, or both. This is especially important before surgery or until immunosuppressive drugs become effective in the face of life-threatening bleeding in immune-mediated thrombocytopenia.[1-3] In the context of platelet transfusion, platelet concentrates (PC) have advantages over whole blood transfusion because of the decreased risk of hemolytic transfusion reactions, volume overload, and the development of polycythemia.
Platelet concentrates from dogs are prepared by serial differential centrifugation of fresh whole blood.[4, 5] or apheresis.[6, 7] Apheresis results in high quality PC, but it requires special equipment and general anesthesia in veterinary practices. Consequently, PC preparation by the serial differential centrifugation of fresh whole blood is more practical in most veterinary clinics.
A protocol for the preparation of PC from dogs by using serial differential centrifugation was first reported in 1993. In the serial differential centrifugation method, PC are harvested by a high-speed centrifugation of platelet-rich plasma (PRP) after a low-speed centrifugation of fresh whole blood. However, it is difficult to resuspend platelet pellets following the high-speed centrifugation caused by platelet activation. Platelet activation during PC preparation contributes to the development of platelet storage lesions and decreased platelet viability.[8-10] It is therefore important to develop a method to minimize platelet activation during the preparation of PC.
Prostaglandin E1 (PGE1) is a reversible inhibitor of platelet activation because of its ability to increase cyclic AMP levels within the platelets.[9, 11] Although early studies showed that the addition of PGE1 to human PRP facilitated the separation, concentration, and resuspension of platelets,[9, 12-14] there have been no studies of the effects of PGE1 on the preparation of PC in dogs. The aim of this study was to investigate the efficacy of adding PGE1 during PC preparation. We estimated the ease of platelet resuspension after high-speed centrifugation of PGE1-treated PC and then confirmed platelet function before and after transfusion, and also evaluated survival of the PGE1-treated platelets after transfusion.
Materials and Methods
The platelet aggregability in PC before transfusion, the survival of the platelets after transfusion, and the platelet reactivity after transfusion were assessed to evaluate the benefits of modifying the PC preparation by adding PGE1. The platelet viability in PC prepared with PGE1 was confirmed by the comparing platelet aggregability before and after PGE1 treatment (n = 5). The survival of the platelets after transfusion was assessed by flow cytometry using a biotin label. The platelets in PC prepared with PGE1 and that in PRP without PGE1 (both n = 5) were labeled with biotin and then were reinfused into the same individuals. Blood samples were collected 1 hour after transfusion (Day 0) and at the same time on Days 1 through 7. The survival time of the transfused platelets were calculated by the changes in the biotin-positive ratio of the platelets. Subsequently, the biotinylated PC prepared with PGE1 (n = 5) were reinfused and blood samples were collected 1 and 24 hours after reinfusion. The P-selectin expression following thrombin stimulation in the biotin-labeled platelets was analyzed by flow cytometry using FITC-labeled anti-P-selectin antibody to estimate the reactivity after transfusion.
Research protocols were approved by the Animal Experiment Committee of Azabu University. Fifteen healthy Beagle dogs, 11 intact males and 4 intact females, 1–12 years of age and weighing 9–15 kg, were used for the experiments. Some dogs were rotated and used for more than 1 experiment (the number of dogs used in each experiment is described below).They were maintained in the Institute of Life Sciences or in the Veterinary Clinical Center, Azabu University, according to the Animal Experiment Guidelines of the university. They had no history of venipuncture or treatment with any drug, including nonsteroidal anti-inflammatory agents, for at least 2 weeks before the study. They were allowed free access to water but were fasted for at least 12 hours before blood collection and were fed after the last sampling was complete on each experimental day.
A quantity of 200 mL of whole blood was drawn into 200 mL blood transfusion bagsa containing citrate-phosphate dextrose adenine 1 (CPDA-1) at the standard mixing ratio (1 part CPDA-1 to 6.5 parts blood) from the jugular vein through 17-gauge needles. The bag was weighed during blood collection, and the collection tubing was clamped off when it reached the intended collection volume.
Platelet concentrates were prepared by serial differential centrifugation of fresh whole blood.[4, 5] All blood components containing platelets were handled gently at 20–24°C (room temperature) to minimize platelet activation. After resting for 30 minutes, the whole blood was centrifuged (soft spin) for 3.5 minutes at 1,000 × g to prepare PRP. The PRP were then transferred into a satellite bagb using a plasma extractor. Extraction was stopped when the red blood cell interface was 1 cm from the top of the collecting bag, and the PRP was left undisturbed for 15 minutes. For PC that were to be treated with PGE1, PGE1c (1 mmol/L in ethanol) was injected into the PRP (final concentration, 1 μmol/L) through an outlet port, and then mixed manually by rocking the bags back and forth. The PRP was centrifuged (hard spin) for 10 minutes at 2,000 × g to produce a platelet pellet. The platelet-poor plasma (PPP) was transferred to another satellite bag, leaving behind 20 mL of plasma with the platelet pellet. The platelet pellet was left undisturbed for 1 hour to promote platelet disaggregation, and then the platelets were resuspended by gentle manual back and forth rocking and massaging of the bag.
Aggregability of Platelets in PC Prepared with PGE1
The aggregability of the platelets in PC treated with and without PGE1 was assessed. Four samples for platelet aggregometry were collected from the transfusion bags at each step of PC preparation. First, a sample of the PRP was collected from the satellite bag when the PRP was produced, and the remainder was divided in 2 satellite bags. Second, the PGE1 was added to one of the separated PRP bags, and then a sample (PGE1+ PRP) was collected immediately. Third, the PGE1+ PRP was centrifuged (hard spin), and then the supernatant was replaced with PGE1-free autologous plasma. The platelet pellet was resuspended after a 1 hour incubation, and a sample of the PGE1-removed PC was collected. Fourth, a control PC sample was also collected from the paired untreated PRP bag after high-speed centrifugation, after a 1-hour incubation, and resuspension according to the reported method.[4, 5]
Aggregometry was performed as follows. First, the platelet counts were measured using an automated cell counterd and adjusted to approximately 250,000/μL by adding autologous PPP. After a 30 minute rest for the resuspended platelets, optical platelet aggregometry was performed by using an aggregometer.e Collagenf and adenosine diphosphate (ADP)g were used as agonists. Because CPDA-1 is not an ideal anticoagulant for aggregometry,[15, 16] the stimulating conditions were modified with the addition of CaCl2 to a final concentration of 15 mmol/L to accelerate the platelet reaction, and the addition of low molecular weight heparinh at a final concentration 0.6 IU anti-Xa/mL to prevent the plasma coagulation induced by adding the CaCl2. The final concentrations of collagen and ADP were 100 μg/mL and 20 μmol/L, respectively.
Survival of Platelets after Transfusion
The survival of the platelets after transfusion in PC prepared with PGE1 and that in PRP without PGE1 were investigated according to Heilmann's technique of biotinylation.[7, 16, 17] The PC and PRP were produced as described above. Next, the platelet survival was assessed as follows: N-hydroxysuccinimido biotin (0.2 mg/mL PC or PRP)i dissolved at 20 mg/mL in dimethylsulphoxide was added into PC prepared with PGE1 or PRP, and then mixed well. After a 10-minute incubation at room temperature, the biotin-treated platelets were infused to the same individuals through a cephalic vein over 30 minutes. Quantities of 2.5 mL of blood samples were collected 1 hour after reinfusion (Day 0) and at the same time on Days 1 to 7 to assess the survival of the reinfused platelets. The samples were anticoagulated with ethylenediaminetetraacetic acid disodium salt and centrifuged for 1 minute at 2,000 × g to separate the PRP. Then the PRP were stored at 4°C until the final sample was collected.
After collecting all the samples for the platelet survival study, the platelet counts of each PRP sample were adjusted to approximately 10,000/μL using a phosphate buffered physiological saline containing 3% bovine serum albumin at pH 7.2 (PBS). A quantity of 10 μL of phycoerythrin-streptavidin (PE-SA)j was added to 100 μL of the platelet suspensions. The percentages of biotinylated platelets were measured by PE positive events using flow cytometry.
The samples were analyzed using an Epics XL flow cytometerk or a Cyflow SL instrument.l The light scatter and fluorescence channels were set on a logarithmic scale. DNA-check beadsm were used to calibrate the fluorescence. The forward scatter threshold was set to exclude debris and machine noise. Platelets were identified by their forward and side light scatter. A minimum of 2,000 platelets were analyzed. The background fluorescence was evaluated by use of nonbiotinylated platelets, and gates were established so that approximately 95% of the events had no fluorescence.
The survival of the platelets after transfusion was calculated using a linear regression model.[18, 19]
Platelet Reactivity after Transfusion
To investigate the platelet reactivity after transfusion, thrombin-induced P-selectin expression was analyzed. The Heilmann platelet biotinylation technique described above was also used to discriminate the transfused platelets from the other platelets.[7, 16, 17] Biotinylated PC prepared with PGE1 were reinfused as described above. A quantity of 2.5 mL of blood was collected into sodium citrate at 1 and 24 hours after reinfusion to assess platelet P-selectin expression with thrombin stimulation.[7, 20, 21]
Because the P-selectin expression changed over time, the analyses of P-selectin expression were performed at 1 and 24 hours after reinfusion. The PRP was separated from these samples as described above. After a 15-minute incubation of the PRP, PGE1 at final concentration of 1 μmol/L was added to the PRP to stop the activation of the platelets. Next, the PRP was centrifuged for 3 minutes at 2,000 × g, and platelets were resuspended in PBS at approximately 10,000/μL. A total of 100 μL of the platelet suspension was incubated with 20 μL of FITC-labeled anti-human P-selectin mouse monoclonal antibody7,21,n and 10 μL of PE-SA.
While they were incubated with the antibody, the platelets were activated with thrombino at a final concentration 0.25 U/mL in the stimulus group. This was done to determine if the transfused platelets maintained their reactivity to thrombin stimulation. After a 20-minute incubation at room temperature and 2 washes, the platelets were resuspended in 150 μL of PBS and analyzed immediately using flow cytometry.
The positive and negative control data were also acquired from preliminary studies where platelets were not transfused, but where they were thrombin stimulated or were left unstimulated.
The FITC-labeled mouse IgG1p was used as an isotypic control for the anti-P-selectin monoclonal antibody, and no nonspecific binding was observed (data not shown). Although the same flow cytometer settings as described above were used, the background fluorescence was evaluated by use of nonstimulated platelets. Compensation was performed for all dual-labeled samples to ensure adequate separation of events with and without fluorescence.
All data were analyzed using the same statistical software program.q Normality was confirmed by the Kolmogorov-Smirnov test. The maximal aggregation values were compared between the PRP and other fractions using Dunnett's posthoc test. The unpaired t-test was used for the analysis of the platelet survival after transfusion. Unpaired t-tests were performed to analyze the differences in the platelet reactivity between the positive control and the values after transfusion. In addition, the paired t-test was used to compare the platelet reactivity between samples collected at 1 and 24 hours after transfusion. Data were expressed as the means ± standard deviation (SD). Box plots were also used as descriptive parameters within figures. Values of P < .05 were considered to be significant.
Platelet Resuspension after High-Speed Centrifugation
Platelets were packed tightly to the bottom, irrespective of the presence of PGE1 (Fig 1A). The platelet pellets prepared with PGE1 achieved uniform resuspension after the 20-minute agitation (Fig 1B). Because the untreated platelet pellet was difficult to disaggregate, there were more platelet aggregates in the bag without PGE1 (Fig 1C). The platelet pellets without PGE1 took 30 minutes or more to achieve uniform resuspension.
Aggregability of Platelets in PC Prepared with PGE1
Although there were significant differences in the aggregability between PRP and PGE1+ PRP (collagen: P < .001; n = 5, ADP: P < .001; n = 5, Fig 2A and 2B), there was no significant difference in the aggregability between PRP and PGE1-removed PC using either agonist. As a result of the replacement of PGE1-containing plasma with intact PPP, the collagen or ADP-induced platelet aggregation recovered similar to the PRP.
Survival of Platelets after Transfusion
Transfused platelets were discriminated as biotin/PE-SA positive events (Fig 3). The platelet survival was 8.04 ± 0.49 days (n = 5) in the group that was transfused with PRP without PGE1 and was 7.56 ± 0.81 days (n = 5) in the group that was transfused with PC prepared with PGE1 (Fig 4). No significant difference was found between the groups in the platelet survival (P = .3).
Platelet Reactivity after Transfusion
Transfused platelets were completely discriminated from other platelets as PE-positive events (Fig 5). There were no significant differences between PE-positive and PE-negative platelets with regard to the P-selectin expression that was evaluated as FITC positive events. Thrombin-induced P-selectin expression events were compared between transfused PC and control data (Fig 6). There were no significant differences between the positive control (P-selectin positive ratio: 53.7 ± 5.1%; n = 5) and transfused PC prepared with PGE1 (1 hour after transfusion: 42.0 ± 13.0%; n = 5, 24 hour after transfusion: 47.9 ± 13.3%; n = 5) by unpaired t-tests. No significant difference was found between the values at 1 and 24 hours after transfusion by the paired t-test.
This study suggests that the addition of PGE1 during preparation of PC by serial differential centrifugation is an effective tool for decreasing platelet activation.
It is often difficult to resuspend platelet pellets following high-speed centrifugation of PRP according to the reported protocol,[4, 5] and treatment with PGE1 made platelet resuspension easier and faster. Therefore, addition of PGE1 resolved the difficulties associated with platelet pellet resuspension in dogs.
To treat hemostatic abnormalities caused by thrombocytopenia, thrombocytopathy, or both, platelets in PGE1-treated PC should act normally after transfusion. The inhibitory effect of PGE1 on platelets is considered to be reversible,[9, 11] but it was necessary to confirm the reversibility in vitro before a PGE1-treated PC transfusion is performed. In this study, the collagen- or ADP-induced platelet aggregation was clearly inhibited by PGE1 treatment. After replacement of the supernatant plasma containing PGE1 with autologous intact PPP, the inhibitory effects of PGE1 were lost. This reversibility of the effect of PGE1 suggested that platelets in PGE1-treated PC act normally during hemostasis when they are diluted with a larger amount of circulating plasma after transfusion.
Whenever new platelet-rich products are produced, an assessment of the in vivo platelet survival is essential to predict the platelet viability after transfusion. We found that the platelet survival after transfusion of PGE1-treated PC was equal to that of intact PRP.
The reversible effect of PGE1 on platelets was confirmed in vitro using platelet aggregometry as described above. However, the viability of platelets after transfusion in PGE1-treated PC is not necessarily guaranteed by evidence of their aggregation. Therefore, we investigated the platelet reactivity after transfusion. Although there are many kinds of laboratory examinations that can be used to assess the quality of PC, there are no in vivo methods to predict the clinical efficacy. For this study, we combined the thrombin-induced P-selectin expression analysis with Heilmann's biotinylation technique[7, 16, 17] which distinguishes transfused platelets from other intact platelets using flow cytometry. This original technique was considered to be quite powerful to estimate the viability of the platelets after transfusion. Our results showed that the strong inhibitory effect of PGE1 on platelet function had disappeared after PC transfusion, and that platelets in PGE1-treated PC maintain their reactivity for at least 24 hours after transfusion.
Because the PGE1 solution is available commercially and is licensed for clinical use, the addition of PGE1 during preparation of PC is easily implemented and practical. In human medicine, the addition of PGE1 to the preparation of PC and 5 days of storage reduced the survival of platelets after transfusion to 5.8 ± 1.6 days compared with 6.9 ± 1.4 days in control PC prepared without PGE1. Although the addition of PGE1 did not shorten the survival of platelets after transfusion in this study, we did not assess the in vivo survival of stored platelets treated with PGE1. Therefore, the addition of PGE1 should only be considered when fresh PC is transfused immediately in dogs. Furthermore studies are needed to investigate the storage stability of PC prepared with PGE1.
This study was partially supported by The Promotion and Mutual Aid Corporation for Private Schools of Japan, and a Grant-in-Aid for Matching Fund Subsidy for Private Universities.
Teruflex, BB-SCD207J01, Terumo Company, Tokyo, Japan
Transfer Bags, BB-T015CJ, Terumo Company
Prostaglandin E1, Sigma Chemical Company, Saint Louis, MO
Sysmex F-820, Sysmex Corporation, Kobe, Japan
Chrono-log C550 dual-channel aggregometer, Chrono-log Corp, Havertown, PA
Chrono-collagen, Chrono-log Corp
Chrono-ADP, Chrono-log Corp
FRAGMINiv5,000, Pfizer Japan Inc, Tokyo, Japan
N-hydroxysuccinimido biotin, Sigma Chemical Company
Phycoerythrin-streptavidin, Sigma Chemical Company
EPICS XL, Beckman Coulter, Fullerton, CA
CyFlow SL, Partec Gmbh, Munster, Germany
DNA-check Beads, Beckman Coulter, Fullerton, CA
Anti-human CD62P-FITC, clone AC1.2, Becton Dickinson, San Jose, CA
Thrombin, Sigma Chemical Company
Mouse IgG1 isotype control-FITC, clone MOPC-21, Becton Dickinson, San Jose, CA
Prism, Version 5.0, Graphpad Software, San Diego, CA