Platelet storage lesion(PSL) is a term used to describechanges seen in platelets during their storage, from the time of their processing until they are transfused to the recipients. Thesechanges reduce the shelf-life or viability of platelets, leading to poor response to transfusion in patients[1]. Storage changes are evident in a host of parameters, such as morphology and biochemical properties, as well as changes due toplatelet activation during processing and storage. These processes initiate a cascade of reactions which lead to the release of granule contents such as P-selectin. P-selectin is a platelet activation markerassociated withincreased adenosine triphosphate (ATP) utilisation; hence, oxygen and glucose levels fall due to activation of glycolysis bythe oxidative pathway.[2]Lactic acid is a by-product of metabolism and acts as an indicator of decreased survival in vivo. Lactic acid accumulation is evident if the pH falls below 6.0 and is associated with poor post-transfusion platelet viability.[3]Platelet surface levels of P-selectin indicate the loss of membrane integrity due to an acidic environment, which leads to swollen and aggregated platelets.[4]Hence, afall in pH, ATP and glucose levels and a rise in lactate dehydrogenase(LDH) levelsin platelet concentratesare indicators of decreased quality of the platelet concentrate. As the platelets undergo changes during storageover 5 days, the number of viable and functional platelets decreases, leading to a decreased platelet count in the platelet concentrate(PC). They also change in morphology from disc-shaped to sphericalanddendritic forms, which increases mean platelet volume(MPV) and platelet distribution width(PDW). Disc-shaped plateletsare of superior quality, indirectly indicating the viability of stored platelets. [4] The platelet concentrate’squality check is done using platelet indices, as these reflect anychange in the morphologyof platelets during storage. The decrease in function and viability can also be determined by the disappearance of the ‘swirling phenomenon’ by visual inspection of stored platelets in thebag. Presence of swirling correlates with platelet function; with increased storage duration, the amount of swirling decreases.Hence, it can be used as a reliable method of quality control.[5] P-selectin released after degranulation can be utilised as a marker to assess in-vitro platelet activation. [6] These parameters indirectly indicatetowards the quality of platelets, and any variation in them would be a measure of the post-transfusion viability of platelets.[7] This study was purported to assess the transformations in platelet morphology, biochemistry and platelet activation markers in platelet concentrates stored for five days at twenty-two degrees Celsius under continuous agitation.

The study was conducted atthe Department of Pathology, Himalayan Institute of Medical Sciences, from December 2015 to August 2017.Informed consent was taken from the donors. The study was conducted in accordance with the Declaration of Helsinki. Clearance was obtained from the institutional ethics committee. (SRHU/HIMS/ETHICS/2017/104) The sample size was calculated using the formula, n = [ (Zα + Zβ) * σ_d ]² / d² n = [ (1.96 + 0.84) * 1.2 ]² / (0.5)² Therefore, n ≈ 45.16 → Round up to 46 The minimum sample sizerequired to detect the specified effect was 46. The study was conducted within a busy clinical transfusion service where a larger number of platelet concentrate units were routinely processed and were available for analysis during the study period. To maximise the robustness and generalizability of the findings, all eligible platelet concentrates were used amounting to 107 available units. Hence, 107 donor platelet concentrateswere assessed on day zero and day five of preparation for swirling, platelet indices (platelet count, mean platelet volume and platelet distribution width), pH, glucose level and morphology. The Platelet Rich Plasma (PRP) method was used to prepare platelet concentrates. This method involves an initial ‘light spin’ followed by a subsequent ‘heavy spin’ in a cryofuge (Thermofischer Scientific) to separate the platelets. Platelets were stored in 450ml quadruple bags(Terumo Penpol SAGM). The presence or absence of swirling was assessed by flipping the bag and observing golden swirls under bright light. Samplesdrawn using an aseptic technique from platelet concentrate bags were used to measure various parameters. The indices were measured by the Beckman Coulter automated analyser (LH750). Glucose levels were measured bythe hexokinase method witha commercial kit(Beckman Coulter),andpH was measured by a digital pH meter. The morphology of plateletswas determined by the modified version of Kunicki score[8], it is scored using the Improved Neubauer’s Chamber. Forty random samples from the total number of samples were assessed for P-selectin levels on day zero and day five of preparation by Enzyme Linked Immunosorbent Assay (ELISA) using a commercial kit(Weldon Biotech Human CD62P ELISA KIT). Samples for ELISA were prepared by centrifuging concentrates and cryopreserving the supernatant in -20 degrees Celsius until the required number of samples were collected. Statistical analysis: The data analysis plan involved using descriptive statistics (mean ± standard deviation) to summarise all parameters. To compare the measurements between day zero and day five for the same platelet concentrates, paired statistical tests were applied; specifically, a paired t-test was used for continuous variables like platelet count, MPV, and pH to determine if the observed changes were statistically significant (with a p-value < 0.05 considered significant). Furthermore, correlation analysis was performed using Pearson's correlation coefficient (r) to assess the strength and direction of the linear relationships between key variables, such as P-selectin levels and morphology scores, with other biochemical and morphological parameters, in order to understand the interconnections within the platelet storage lesion. All analyses were conducted using the Statistical Package for the Social Sciences (SPSS) version

In this study, all the random donor platelet bags (n=107)(100%) showed the presence of swirling on day 0, while it was absent in 10% bags on day 5. The mean platelet count of the platelet concentrates decreased from 679±226.46 x103/µL on day zero to 418.18±212.62 x103/µL on day five (p <0.01). The platelet concentrates had an average mean platelet volume (MPV) of 7.30±1.07fL on day zero, which increased to 8.59±1.23fL on day five (p less than 0.01). The average PDW of the platelet concentrates on day zero was 17.66±0.64, which increased to 18.47±0.69 on day five (p<0.01). The average Kunicki Morphology Score of the platelet concentrates on day zero was 183.93±5.56, which decreased to 160.67±10.45 (p-value is less than 0.01). The calculated mean pH of platelet concentrates on day zero was 7.97±0.41, which decreased to 6.79±0.33 on day five (p<0.01). The mean glucose concentration of the platelet concentrates on day zero was 377.76±59.09 mg/dL, which decreased to 264.31±84.35 mg/dL on day five (p<0.01). The average p-selectin optical density in platelet concentrates (n=40) on day zero was 2.301±0.63, which increased to 2.677±0.451 on day five. (Table 1) P-selectin showed a positive correlation withMPV and PDW, indicating that with an increase in P-selectin, an increase in these parameters also occurs. P-selectin and glucose levels showed a negative correlation. No correlation was found between P-selectin and pH levels. A positive correlation was observedbetween morphology score andplatelet count, pH and glucose level, indicating that an increase in morphology scoreis associated with subsequent increases in these parameters. A significant negative correlation was observed between the morphology score andMPV and PDW, implying that a decrease in the morphology score is associated with a subsequent decrease in these parameters. (Table 2) TABLES: Table 1: The mean values of various parameters of platelet concentrates observed on day zero and day five. Parameter (n=107) Day Zero Day Five Swirling Present = 100% Absent = 0% Present = 90% Absent = 10% Platelet Count (x103µL) 679±226.4 418.1±212.64 Mean Platelet Volume (fL) 7.30±1.07 8.59±1.23 Platelet Distribution Width 17.66±0.64 18.47±0.69 Kunicki Morphology Score 184±5.56 160±10.45 pH 7.97±0.41 6.79±0.33 Glucose (mg/dL) 377.76±59.09 264.31±84.35 P-selectin (n=40) 2.301±0.63 2.677±0.451 Table 2: Correlation of P-selectin levels and Morphology Score with other parameters Parameters Pearson Correlation (P-selectin) Sig (2-tailed) Pearson Correlation (Morphology Score) Sig (2-tailed) Morphology Score -0.241 0.31 - - MPV 0.228 0.042 -0.438 0.00 PDW 0.370 0.001 -0.510 0.00 pH 0.196 0.82 0.697 0.00 Glucose -0.356 0.001 0.486 0.00 Platelet Count - - 0.433 0.00 Correlation is significant at p-value <0.05

Many studies have been done in the past decades on the changes occurring in the platelet concentrates stored for 5-7 days. These studies measured various parameters by different methods to determine these changes taking place over each day. The aim of this study was to detect the changes taking place in the biochemical parametersof the supernatant plasma of 107 platelet concentrates, along with the changes in platelet indices and morphology. On gross examination, swirling is a reliable indicator for quality assessment of platelet bags.[9] The bending or diffraction of light causes platelet swirling with normal discoid-shaped platelets, and with shape change, the amount of swirling decreases.[10] Swirling was observed in all the bags on day zero of preparation, which decreased to 90% of the bags on day five. This finding is consistent with similar studiesconducted by A Guptaand S Bashir. [11][12]This indicates that swirling as a parameter for measuring platelet quality is reasonably reliable. The effectiveness of measures taken to prolong storage duration and maintain the quality of platelets can also be determined by the presence of swirling. The haematoanalyzercalculates platelet indices, which are derived parameters obtained from a complete blood count. Amongst all parameters, MPV and PDW are directly related to platelet morphology.[13] Platelet count of the PCsdependson the whole blood count of the donor and thestorage duration, affecting the post-transfusion increment in the platelet count. In this study, the mean platelet count decreased over five days of storage. A M Albanyan, S F Ali and their colleagues also observed a significant decrease in platelet count similar to ours on the seventh and fifth days of storage, respectively, whereas Singh et al found no significant difference. [14][15] Residual platelet function in stored plateletsis reflected by the MPV,its increaseindicates deterioration. However, the acceptability criterion for it is still unclear. MPV of plateletsincreased duringfive days of storagewhich was also observed byBashir et aland Gulliksson et al in their studies. [12][16]It is assumed that the rise in MPV occurs partially due to the swelling of the platelet and partially due to the formation of the dendritic processes caused by platelet activation. A standard haematology cell counter is used to assess the PDW, which indicates the difference in sizeof the platelets or anisocytosis and provides an adjunctive measure of quality.[17] In the present study, the average PDW increased over 5 days. Albanyan et alobserved differences in PDW of both buffy-coated platelets (BC-P) and Apheresis platelets (AP-P)during storage.On one hand,asizeable increasein PDW of AP-P was observed;no change in the mean PDW of BC-P was observed, which was discordant with our study.[14]Ray et al observed that nearly 93% samples showed an increase in the PDW and only 71% showed an increase in the MPV, clearly showing that the PDW is a better marker of quality control than MPV for both processing and storage.[17] These parameters indicate a change in the morphology of platelets. There was a significant correlation between the morphology score and PDW (r=-0.510) and MPV (r=-0.438).As PDW changes with platelet activationand reflects the change in platelet morphology, it may indirectly contribute to an increase in storage lesions.[13] Althoughfurther studies might be needed to reach conclusive evidence. The change in platelet morphology is said to be the most dependable in-vitro determinant for the post-transfusion survival of platelets. [18]A modified versionof Kunicki score was used to grade the morphological changes taking place in this study, which revealed a significant decrease in the average score from 183 to 169 over five days. In a study similar to ours, conducted by Jain et al, the Modified Kunicki scoringfrom 192 to 142 over five days of storage.[19] In addition, they also found a significant correlation between morphology and P-selectin levels (r=-0.351) and morphology score and pH levels, which was concordant with our correlations with pH and P-selectin. Rock et alalso had similar observations regarding morphology and its correlation with platelet activation.[20] The pH level is also an establishedparameter to ascertain the quality of preserved platelet concentrates, and a pH below six is associated with a significant decline in post-transfusion survival of platelets.[21] The pH decreases during storage due to lactate production during glycolysis.[22]Furthermore, MPV progressively rises, which points to swelling and accelerated change from normal discshape to a spherical shape.[23] A significant decrease was observed in the pH from 7.9 to 6.7 over five days of storage at room temperature under constant agitation. Jain et al also observed pH became acidic during the storage of platelet concentrates.[19]However, Ali et al observed that during storage for up to five days, buffy-coated platelet concentrate (BC-PC) units displayed no significant difference in pH,whileplatelet-rich plasma (PRP) PCsshowed a fall in pH.[15]A small population of platelets in the concentratesmight have an enhanced abilityfor lactate formation, whichcan cause a significant reduction in pH during storage of many platelet concentrates.[24] Glucose level is another biochemical parameter, the measure of which is also indicative of Platelet Storage Lesion. The change in glucose level occurs due to cellular metabolism, which leads to lactate production, which makes the environment acidic. The average glucose levels declined over five days from 377mg/dL to 264mg/dL. Jain et al also noted a significant decrease in glucose levels from 380mg/dL to 320mg/dL over 7 days.[17] Gupta et al had similar results concordant with our study. [11] Presence of P-selectin and glucose levels showed a negative correlation(r=-0.356). This might occur due to the utilisation of glucose for platelet activation, leading to increased p-selectin. So, neitheran excessivenor a minusculeamount of glucose is adequate for optimal storage of platelet concentrates. Glucose is utilised byplatelets as a source of energy, and when it lowers further, the storage changes start to occur. Adding glucose to storage solution to platelet concentrates might increase the longevity of PCs, but it should be done within the acceptable limits. P-selectin (CD62P) is a platelet activation-specific surface antigen. There was a significant rise in P-selectin levels over five days of storage. Gulliksson et al also observed a similar increase in P-selectin after five days of storage. Although hemeasured the surface CD62P by flow cytometry, the result is comparable to our study in which ELISA was used to quantify soluble P-selectin levels, which increased over time. [22] The increase in P-selectin being detrimental to platelet function is debatable. It is proven that there is continuous release of P-selectin from the a-granules until they are completely exhausted, but factors such as the method of platelet concentrate preparation and storage conditions also affect the extent to which P-selectin is produced. Hence, platelet storage lesions(PSLs) can be assessed by measuring various biochemical, morphological and molecular parameters of platelet concentrates.a)Swirling – which is the easiest, non-invasive method for quality assessment and a gross measurement of the microscopic morphology change. However, its limitation is inter-observer variation. b) Morphology – which can be assessed directly by microscopic methods or indirect assessment via platelet indices. c) Platelet indices (Platelet count, MPV, PDW) – the number of platelets present in the platelet concentrate and measures of their variability help not only in determining the extent of storage lesion but also help to assess the post-transfusion increase in platelet level. d) Biochemical parameters – Glucose and pH are indirect measures of platelet activation. e) Lastly, P-selectin, which is a known marker of platelet activation, can be used to assess PSL, whether it is surface CD62P or its soluble form. Numerous factors influence the quality of platelet concentrates. One should take each one of them into careful consideration to devise techniques to increase the longevity of stored platelet concentrates from the current 5 days to 10days. Exploratory studies need to be done on platelet additive solutions (PAS) and lower storage temperatures to improve shelf-life of platelet. There is an inherent risk of microbial contamination of platelet concentrates when stored at room temperature(21-22 degrees Celsius). Microbial contamination duringstorageis another parameter which can be assessed in future studies.Studies can also be conducted on the influence of different methods of PC preparation on the shelf-life of platelets.

To conclude, this studyreiterates findings from the literature that significant changes occur in various parameters of stored platelet concentrates over 5 days. There is an absence of swirling after prolonged platelet storage, and a decrease occurs in platelet count, morphology score, pH and glucose levels of platelet concentrates in storage, while an increase in mean platelet volume, platelet distribution width, and p-selectin levels also occurs during storage of platelet concentrates. With prolonged storage, there is activation and a change in shape of platelets from discoid to swollen and dendritic forms, as demonstrated by the change in morphology score and p-selectin levels showing significant correlation. In conclusion, all these parameters are reliable markers to determine the extent of platelet storage lesion. Various factors affect the changes taking place in these parameters during storage which should also be taken into consideration. However, further studies might be needed to delve deep into the mechanics of platelet storage changes and increase the shelf life of stored platelet concentrates.

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