Influence of Different Methods Preparation on Platelet Activation in Stored Platelet Concentrates

Platelet transfusion therapy has played an important role in the management of patients [4]. Today, a variety of methods for the preparation of platelets for transfusion are available ,which may be due to different processing systems such as apheresis ,buffy coat and platelet rich plasma platelet concentres methods [5,6]. Processing and storage may affect both platelet quality and function and they may thus be less effective when transfused than fresh platelets [7-9]. Platelet functional assessment can be performed by a number of different methods; including cell counting, pH and markers for platelet activation.


Introduction
Blood banks optimize their protocols to yield the highest quality products in the shortest possible time. Optimized protocols may vary from blood bank to blood bank depending on which blood products are most desired. The issue of platelet quality during extended storage has been well addressed through studies using a variety of in vitro measures [1][2][3].
Platelet transfusion therapy has played an important role in the management of patients [4]. Today, a variety of methods for the preparation of platelets for transfusion are available ,which may be due to different processing systems such as apheresis ,buffy coat and platelet rich plasma platelet concentres methods [5,6]. Processing and storage may affect both platelet quality and function and they may thus be less effective when transfused than fresh platelets [7][8][9]. Platelet functional assessment can be performed by a number of different methods; including cell counting, pH and markers for platelet activation.
In the laboratory, phosphatidylserine exposure and its subsequent procoagulant activity can be assessed by several methods including flow cytometry utilising the binding of fluorescently labelled Annexin V. The final stage of platelet activation is the capacity of the platelet to expose phosphatidylserine [10][11][12].
Annexin V was used as a parameter for quality monitoring of platelet concentrates during storage. Activation of platelet is followed by an increased CD63 expression; this protein secreted from the granules during storage [2,[12][13][14]. Lactatedehydrogenase (LDH), a marker of disintegration of platelets as a parameter for activation marker of platelet concentrates during storage.
In summary this thesis has demonstrated assays to detect the quality in the platelet derived by different methods.

Platelet preparation
Preparation of buffy coat-platelet concentrate (BC): Twenty five whole blood was collected in a 450 ml quadruple bag containing 63 ml of CPDA1 anticoagulant (TERUMO PENPOL, Ltd., Puliyarakonam, Trivandrum, India). The whole blood was first subjected to "hard spin" centrifugation at 3640 rpm for 7 minutes at 22°C. After centrifugation the supernatant plasma and the subnanat red cells were transferred into attached satellite containers. Platelet poor supernatant was expressed into one satellite bag and buffy coat into another satellite bag. About 25-40 ml of plasma was returned to buffy coat with the aim of cleaning the tubing from residual cells and obtaining an appropriate amount of plasma in the BC. The buffy coat was gently mixed with the plasma and again subjected to 'light spin" centrifugation at 1180 rpm for 8 minutes at 22°C, along with one empty satellite bag. Briefly, the main difference lies in the amount of force applied in the first centrifugation step. In the buffy-coat method, a higher gravitational (g) force is applied causing the platelets and leukocytes to form a tight buffy-coat layer between the red cell and plasma layers suspended in the plasma.

Preparation of platelet rich plasma-platelet concentrates (PC):
Four hundred and fifty ml of whole blood was collected in triple bag containing CPDA1 anticoagulant (JMS Singapore Pte Ltd) and PCs was prepared within 4 hours of collection stored for 5 days at 20-24°C under constant agitation (n=25).

Aphaeresis-derived platelet concentrates (APC):
Twenty five healthy volunteer donors each were subjected to this study by Haemonetics MCS plus cell separator. The apheresis sets used for the cell separator was REF 995E. This procedure was done in a closed system. During the procedures, the blood was anticoagulant at the point of withdrawal, and the ratio of whole blood and anticoagulant (ACD) was maintained at 9:1 to 11:1.
At the end of the collection procedure, the platelet collection bag was shaken vigorously to detach the platelets from the wall of the bag and kept for 1 hour at room temperature to make it an even suspension. The final volume of the APC ranged from 250-280 ml.

Laboratory analysis
On days 0, 1, 3, 5 samples from PC, BC and APC were taken for analysis under sterile condition. Manual cell counts were therefore carried out utilizing the improved Neubauer chamber for counting leukocytes (WBC) and platelets count. The pH was evaluated at the end of the day of storage. PH values were determined by the pH meter (Mettler Toledo, witzerland). LDH was determined using colorimetric methods by Roche Hitachi 902 Chemistry Analyzer (Roche, Germany). Using flow cytomerty, we investigated platelet membrane binding of Annexin V (Annexin V Fluorescein-FITC/ApoScreen)and CD63 (Mouse Monclonal Antibody to human CD63-FITC/Caltage) expression in platelet stored for up to 5 days under standard blood banking conditions. Special monoclonal antibodies that conjugated with fluorescence dye in flow cytometric method was used for its.

Statistical analysis
All data were expressed as mean ± SD. We performed statistical comparison by using 't'-test. A probability of P<0.05 (two-sided) was used to reject null hypothesis.

Results
In this experimental study, the mean platelet count of BCs, PCs and APCs was 5.7+1.9 × 10 10 /unit, 5.8+2.1 × 10 10 /unit and 2.4+2.2 × 10 11 / unit. The mean platelet count PCs, BCs and APCs was comparable and statistically significant difference was observed (P<0.05). The volume of individual units was calculated .The mean volume of the PCs, BCs and APCs was 55.6+12.3 ml, 69+20.6 ml and 245+15.6 ml. The mean WBC count in PC, BC and APCs units was 41+0.48 × 10 6 /unit, 23+0.49 × 10 6 /unit and 3.3+0.8 × 10 6 /unit. The mean leukocyte counts BCs, PCs and APCs was comparable and statistically significant difference was observed (p<0.05). All units of platelet concentrates product met the recommended quality control parameters of volume. LDH and cell count. Apheresis product by Haemonetics MCS plus showed better leucoreduction than BCs and PCs product. Their mean pH was 6.9+0.21 (mean+SD) and ranged from 6.8-7.01 and no difference was observed between all three types of platelet concentrate. The results of pH and cell counts are listed in Table 1.
The mean levels CD63 and percentage of Annexin V was comparable and statistically no significant difference was observed in three types of platelet concentrate on days (0, 1, 3), but there was statistically relevant increase in CD63 and Annexin V on day 5. During storage for up to 5 days PCs units displayed a significant increase in the CD63, Annexin V expressions, as compared with BCs units and APCs units (p<0.05). The mean LDH was comparable and statistically no significant difference was observed in three types of platelet concentrate on days (0, 1, 3), but there was statistically increase in LDH on day 5. The data showed that the level of LDH was significantly in the BCs units and APCs units than that PCs in the days 5 of storage (P-value<0.05). The platelet activation markers CD63, LDH and Annexin V are presented in Table 2.

Discussion
We found statistically significant differences in a variety of cellular, functional and flow cytometry parameters [15]. This study has determined in vitro quality of 5 days stored platelet concentrates prepared by three different methods. The detection of platelet activation has been used as a guide to the assessment of the suitability of techniques or conditions under which platelets have been prepared for transfusion. The present paper describes an in vitro comparison of different preparations. The studies suggested that the degree of in vitro activation as evidenced by the expression of CD63 and Annexin   [2,4,12,14].
We found that the extent of activation was significantly higher in PCs than in BCs and APCs units. It was concluded that, immediately after preparation, PCs are more activated than BCs and APCs units. This is most likely a result of the pelleting that follows the second highspeed centrifugation of the PCs.
However, the different CD63 and Annexin V in BCs and APCs clearly demonstrate that process of activation exceeds that of PCs and rises progressively during storage under blood bank conditions. Although in three preparations, platelet activation is increased by storage time, APCs are characterized by a much better in vitro than PCs [3,5,7]. However, the different CD63 and Annexin V in PCs and APCs clearly demonstrate that process of activation exceeds that of APCs. Platelets processed by the buffy coat technique showed less CD63 expression than platelets prepared by the PCs method. This study shows that in addition to pH, volume and cell counts; in vitro platelet activation markers were used to monitor platelet [3,13,14]. These data suggest that measurement of CD63, Annexin V and LDH may be a more desirable markers for clinical studies of activated platelets, since it may be less susceptible to artifactual elevation due to minor variations in sample handling and assay [3,12,14]. The results of this study showed that the LDH levels were also increased during storage in platelet concentrates. Although LDH level was increased during the storage in the three methods, but the rise of LDH was significantly higher in PCs than that of Bc and APC methods. This study could imply the effects kind of methods for preparation on quality product. The production processes subject the platelets to different kinds of stresses and stimuli. The platelets produced by either apheresis or buffy-coat production methods show little evidence of activation than platelet concentrates (PCs) [16].
Although the ability of transfused platelet to circulate and function is deponent on both the effect of the ex-vivo storage lesions that undermines platelet functionality and the status of the in vivo milieu of the transfused individual.