|Year : 2019 | Volume
| Issue : 1 | Page : 21-27
Comparative analysis of platelet storage lesion in whole-blood-derived platelets stored in autologous plasma versus platelets stored in platelet additive solution using flow cytometric assay of CD62 and Annexin V
Subhashish Das, M L Harendra Kumar
Department of Pathology, Sri Devaraj Urs Medical College, Kolar, Karnataka, India
|Date of Web Publication||22-Apr-2019|
Dr. Subhashish Das
Department of Pathology, Sri Devaraj Urs Medical College, Kolar, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Platelets storage lesions remain a challenge in regular blood transfusion services. Flow cytometric study based on the expression of platelets activation markers in the stored platelets is a good method to study such lesions. Materials and Methods: Buffy-coat-derived platelet concentrates were stored under standard conditions for 7 days. The expression of activation antigens CD62p and Annexin V on total platelets and populations of small, medium-sized and large platelets was analyzed by flow cytometry during storage on days 0, 5 and 7. Results: There was significant difference in Annexin V expression between PAS and non- PAS group on Day 0, Day 5 and Day 7. The same observation was noted with regard to CD62 estimations. Conclusion: Our study confirm the beneficial effects of platelet additive solution as highlighted by the flow cytometric assay of CD62 and Annexin V. Evaluation of the level of expression of various activation markers on different platelet populations could be an additional valid tool in quality control of platelet concentrates, and could be a novel approach towards better platelet inventory management.
Keywords: Annexin V, CD62, flow cytometry, platelets, platelet storage lesion
|How to cite this article:|
Das S, Kumar M L. Comparative analysis of platelet storage lesion in whole-blood-derived platelets stored in autologous plasma versus platelets stored in platelet additive solution using flow cytometric assay of CD62 and Annexin V. Glob J Transfus Med 2019;4:21-7
|How to cite this URL:|
Das S, Kumar M L. Comparative analysis of platelet storage lesion in whole-blood-derived platelets stored in autologous plasma versus platelets stored in platelet additive solution using flow cytometric assay of CD62 and Annexin V. Glob J Transfus Med [serial online] 2019 [cited 2019 Sep 20];4:21-7. Available from: http://www.gjtmonline.com/text.asp?2019/4/1/21/256741
| Introduction|| |
Blood transfusion is a life-saving intervention and refers to the use of blood and its components for therapy. There is an ever-increasing need and demand for blood as transfusion supports various forms of medical care such as fetal and obstetric care, surgery, and trauma and in the treatment of heart ailments, cancer, and degenerative conditions. The main principles of transfusion lie in safe and effective blood supply. The paradigm shift in blood transfusion toward the “safety” of the transfused blood is in accordance with the ever-changing techniques of storage of whole blood and its components.
In this study, whole-blood-derived platelets were labeled with fluorescent monoclonal antibodies – CD62 and annexin V, and level of expression was studied using flow cytometry. Further, CD62 and annexin V expression was studied in platelets stored in autologous plasma versus platelets stored in platelet additive solution (PAS). The principle of the test is that the intensity of the emitted light is directly proportional to the number of antibodies attached to the platelet receptors/antigens which get attached upon activation.
Processing of the unfixed sample immediately after collection is the preferred method for flow cytometry. However, as the sample had to be shifted to another center for testing, the platelet was fixed in paraformaldehyde before transportation and testing. Immediate fixation is preferable for the study of time-dependent changes in platelet activation.
CD62P is a membrane glycoprotein in alpha-granules of platelets. On activation of platelets, exocytosis of the alpha-granules leads to CD62P exposure on the platelet surface. The expression of CD62P on the platelet surface reflects the degranulation of platelets and thus serves as a moderately sensitive marker for the activation status of platelets in vitro and in vivo.,
During preparation and subsequent storage, platelet concentrates become gradually activated and partially lose their membrane integrity, leading to the so-called platelet storage lesion (PSL). In case of PSL, there is an increased expression of CD62 on the surface of platelets, and annexin V is expressed during apoptosis. Annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine.
| Materials and Methods|| |
This was a prospective comparative study of PSL in platelets stored in autologous plasma versus platelets stored in PAS done at our blood center.
- Whole blood was collected in quadruple bags from a total of 156 voluntary and replacement donors between January 2018 and June 2018. Platelets were prepared by the buffy coat method and stored in plasma or PAS and analyzed for CD62 and annexin V. Standard donor selection criteria in line with NACO/NBTC guidelines were used. A total of 156 samples were randomly selected and divided into two groups. One group of 78 was stored in plasma and another with an equal number was stored in PAS. Only those donors with a platelet count between 150 × 103/μl and 250 × 103/μl were enrolled in the study
- A small quantity (2 ml) of platelets was collected from freshly prepared platelet bag for testing by flow cytometry, by spiking on day 5, and repeated again on day 7. In addition, annexin V expression was estimated on day 0 (day of collection). The bag was stored on platelet agitator in the intervening period. The platelets were fixed using 1% paraformaldehyde before transport and before binding with monoclonal antibodies for flow cytometric assessment, which was done at the referral laboratory.
In the flow cytometer, the suspended cells labeled with the monoclonal antibodies and tagged with fluorescent dye were passed through the flow chamber, at a rate of 1000–10,000 cells/min, through the focused beam of a laser. After fluorescent activation of the fluorophore at the excitation wavelength, a detector processes the emitted fluorescence and light-scattering properties of each cell are captured.
Sodium citrate is the anticoagulant of choice for platelet flow cytometry analysis. Whole blood was collected in citrate-phosphate-adenine bags for separation of platelet concentrates.
A titration study was performed for CD62 and annexin V using four different concentrations (1:50, 1:100, 1:200, and 1:300) to determine the optimal dilution.
Before staining the cells, serial dilutions of the platelet concentrates were made with 0.9% NaCl to achieve a final concentration of 5 × 106 cells/mL in 5-mL round-bottom polystyrene tubes for flow cytometry (Falcon™, Thermo Fisher Scientific, Whitby, ON, Canada).
For preparation of CD62
Once the final concentration of 5 × 106 cells/mL was achieved, cells were washed twice with an aqueous buffered solution containing fetal bovine serum and sodium azide (BD Pharmingen™ Stain Buffer FBS, San Diego, CA, USA), followed by centrifuging the sample (Sorvall™ ST16 Centrifuge, Thermo Fisher Scientific, Waltham, Massachusetts, USA) at 300 g for 8 min. After discarding the supernatant, platelets were stained with 1 μl of a monoclonal mouse anti-human antibody at a final concentration of 1:100 (P-selectin/CD62P Antibody (AK-6) [Alexa Fluor (R) 405] NBP2-22046AF405, Novus Biologicals Canada), gently homogenized with a vortex mixer (Vortex Genie 2, Thermo Fisher Scientific, Whitby, ON, Canada), and incubated at room temperature for 40 min in the dark. Two more washes were made with stain buffer, and samples were analyzed within 1 h of labeling. The samples were protected from light at all times.
For preparation of Annexin V
One milliliter of sample with 5 × 106 cells was poured into a 5-mL round-bottom polystyrene tube for flow cytometry (Falcon™, Thermo Fisher Scientific, Whitby, ON, Canada). The cells were washed twice with a phosphate-buffered saline solution, followed by centrifugation (Sorvall™ ST16 Centrifuge, Thermo Fisher Scientific, Waltham, Massachusetts, USA) at 300 g for 8 min. After discarding the supernatant, platelets were reconstituted with 1 mL of binding buffer (Annexin V Binding Buffer, 10X concentrate, BD Pharmingen™ Becton, Dickinson and Company, NJ, USA) and gently mixed. Subsequently, 100 μL was transferred to a 5-mL culture tube, stained with 1 μL of annexin V (FITC Annexin V Apoptosis Detection Kit I, BD Pharmingen™ Becton, Dickinson and Company, NJ, USA), and gently mixed (Vortex Genie 2, Thermo Fisher Scientific, Whitby, ON, Canada). Cells were incubated at room temperature in the dark for 15 min. The sample was analyzed within 30 min of staining. The samples were protected from light at all times.
Positive control samples for both annexin V and P-selectin were made by activating the platelets with bovine thrombin (Thrombin from bovine plasma, Sigma-Aldrich, St Louis, MO, USA) at a final concentration of 0.3 U/mL in conjunction with glycyl-L-prolyl-L-arginyl-L-proline acetate (GPRP, Sigma-Aldrich, St Louis, MO, USA) at a final concentration of 1 ml, to prevent fibrin polymerization. After the washes and before staining the cells, 2 μL of GPRP was added to the sample and incubated in the dark for 2 min under constant agitation, followed by the addition of 2 μL of thrombin, incubating the sample for 5 min in the dark with constant gentle agitation.
| Results|| |
There was a significant difference in annexin V between PAS group and non-PAS group on day 0, day 5, and day 7 [Table 1]. Annexin V was 7.6% on day 0, 18.4% on day 5, and 23.1% on day 7 in PAS group. Annexin V was 11% on day 1, 22.3% on day 5, and 30.8% on day 7 in without PAS group.
|Table 1: Annexin V comparison between platelet additive solution group and without platelet additive solution group on day 0, day 5, and day 7|
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[Table 2] shows the various subcategories of platelets, namely small, medium, and large on the basis of their size, i.e., forward scatter properties. The storage time-dependent decrease, in the number of large platelets, is significant for both PAS and non-PAS category confirming the in vitro aging of platelets along with fragmentation into platelet subpopulation that acts as a marker for the PSL. However, the decrease is of much lesser degree in the platelets stored in PAS.
|Table 2: CD62 between platelet additive solution group and without platelet additive solution group on day 5 and day 7|
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Flow cytometry evaluation of platelet activation with and without PAS leading to the development of PSL is very helpful in our understanding of the pathogenesis of PSL.
Flow cytometry identification and evaluation of platelet subpopulation including the small medium and large platelets will help in the correct monitoring of the storage lesion and can act as an additional quality control indicator with regard to platelet storage and metabolism.
All statistical analysis and graph generation were performed using GraphPad Prism 7 (GraphPad Software Inc., La Jolla, California, USA).
| Discussion|| |
The advantages of flow cytometry are manifold. Platelets were analyzed in their physiological state. Platelet activation is insignificant during testing as there is minimum manipulation of the sample during flow cytometry. Further, flow cytometry is a highly sensitive and specific procedure for the detection of resting and activated platelets and platelet-derived microparticles.
The following observations/inferences were drawn from our study:
Reduced expression of annexin V and reduction in activated platelet granules (CD62) in PAS platelets as compared to platelet concentrates stored in plasma show that PAS reduces the risk of development of PSL in whole-blood-derived platelets as compared to platelets stored in plasma.
This is possibly due to the acetate in PAS, which is metabolized to bicarbonate preventing the fall in pH that occurs when platelets are stored in plasma. The presence of magnesium (Mg2+) and potassium (K +) in PAS also prevents the lowering of pH and reduces spontaneous activation of platelets during storage and thereby reducing the development of PSL.
Similar to the study conducted by Vučetić et al., our study shows that expression of platelet activation markers correlates with platelet activation parameters detected by standard laboratory tests, confirming that flow cytometry can be used as an additional method for the quality control of PC.
According to Garraud et al., there are 5 million combinations of individual parameters that can influence PC-BC quality. It is, therefore, very difficult to compare our results directly with those of other research groups, and we think that each blood transfusion center should establish its own “in-house” reference range for the assessment of PSL.
Similar to the studies conducted by Vučetić et al., the results of our study shows that mean fluorescence index (MFI) values for all analyzed surface molecules were as follows:
Small platelets < medium platelets < large platelets.
It has been suggested that MFI values could be expressed as a normalized fluorescence ratio to volume to eliminate the influence of platelet size on the overall fluorescence intensity.
The various levels of activation as detected on different days were small > medium > large platelets, clearly proving that maximum activation and subsequent expression were detected on large platelets on day 5 and day 7 of storage. However, PAS solution across all the days helped in minimizing the development of PSL [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6].
|Figure 1: Bar diagram showing annexin V comparison between platelet additive solution group and without platelet additive solution group on day 1, day 5, and day 7|
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|Figure 2: Bar diagram showing CD62 P1 between platelet additive solution and without platelet additive solution: Platelets with platelet additive solution show lesser degree of activation along with aging with fragmentation having lower risk of platelet storage lesion|
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|Figure 3: Bar diagram showing CD62 P2 between platelet additive solution and without platelet additive solution: Platelets with platelet additive solution show lesser degree of activation along with aging with fragmentation having lower risk of platelet storage lesion|
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|Figure 4: Bar diagram showing CD62 P4 between platelet additive solution and without platelet additive solution: Platelets with platelet additive solution show lesser degree of activation along with aging with fragmentation having lower risk of platelet storage lesion|
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| Conclusions|| |
Platelets have a natural life span of 8–12 days. However, the life span of stored platelets collected for therapeutic or prophylactic transfusion is currently limited to 5–7 days at 22°C–24°C, with agitation. This limitation is to attenuate the risk of bacterial growth and diminish the effects of PSL.
Our study shows the difference in CD62 and annexin V expression between PAS platelets and non-PAS platelets stored in plasma. PAS is extremely beneficial in extending the shelf life beyond 5–7 days. This is highlighted by the fact that the degree of platelet activation of platelet concentrates in PAS solution is much lower.
However, more flow cytometry studies need to be conducted to substantiate our findings on a larger scale.
The author sincerely acknowledges the help rendered by the Indian Institute of Science, Bengaluru (IISc), in conducting the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]