Evaluation of Platelet Storage Lesion (PSL) Markers in SSP+ Platelet Additive Solution Added Canine Platelet Concentrates (CPC)

Thrombocytopenia (mild, moderate and severe), a quantitative disorder of reduction in number of circulating platelets is the most commonly occurring haematological disorders in small animal medicine (Hux and Martin, 2012).
       
Severe thrombocytopenia (Platelets < 25,000 cells/µl) due to haemoprotozoal and rickettsial diseases with clinical signs of haemostatic abnormalities are highly prevalent in dogs with an incidence of 32.2% which were presented to the Critical Care Unit, MVC, Chennai and the TANUVAS Animal Blood Bank, MVC, Chennai (Baranidharan, 2015).
       
Conventional fresh CPC stored at 22°C showed optimal post transfusion platelet recovery and platelet functions when compared to chilled platelets, cryopreserved platelets or lyophilised platelets that can be used in severely thrombocytopenic dogs to provide immediate and short-term haemostasis.
       
Globally, CPC transfusions are seldom practiced on account of unavailability of stored platelets due to its short period of storage (5 days) and lack of donors at emergency crisis which are shortcomings that are encountered in veterinary blood banks (Callan et al., 2009). Hence, in this study it was hypothesised that the addition of SSP+ PAS to the CPC stored at 22°C under continuous agitation would increase the storage life and quality of canine PC.
       
Henceforth, the objective of this study was to increase the shelf life of CPC by the use of SSP+ PAS.

The study was conducted at the TANUVAS Animal Blood Bank (TABB) facility at Madras Veterinary College and Teaching Hospital, TANUVAS, Chennai during the period from April 2020 to February 2021. CPC was prepared from eligible donor dogs (n = 6) brought to the TANUVAS Animal Blood Bank of the Madras Veterinary College. Jugular phlebotomy and blood collection was performed using 350 ml Quadruple blood bag system¹ as per standard protocols. The tubing was then sealed was sealed using an automated tube sealer² and the blood bag was allowed to rest at ambient temp (22-24°C) for one hour until further processing. The blood bag was then centrifuged at 3400 rpm for 11 minutes at 22°C in a refrigerated centrifuge³ and an automated component separator⁴ was used to extract the Packed Red Blood Cells into the SAGM containing satellite bag and Plasma component into another satellite bag. Buffy Coat layer was suspended in the bag for at least 2 hours at room temperature which was further centrifuged at 900 rpm for 6 minutes at 17°C and the supernatant PC was extracted into the platelet storage satellite bag. CPC was kept at room temperature for 30 minutes before storing it in the Platelet Agitator⁵ at 22°C.
       
The total amount of CPC was divided into four aliquots of 15 ml to which SSP+ was added at concentrations of 65%, 75% and 85% of the total final volume (Table 1).
 

       
Swirling was assessed for the CPC by holding the bags against a source of bright light and gently agitating the bag. The intensity of the white turbulent cloudy appearance was graded as nil (No swirling), + (mild), ++ (moderate) and +++ (Extensive).
       
The PSL markers were evaluated using a portable arterial blood gas analyses machine⁶ while the platelet indices like MPV and PDW were measured using a auto-haemoanalyser⁷. All samples were studied for the above parameters on days 1, 5, 9 and 13 during storage and were evaluated for bacterial contamination.


 
Statistical analysis
 
The level of swirling was assigned ranks and statistical analysis was done using Kruskal Wallis analysis (Table 2). The statistical analysis was performed using SPSS software. Two Way ANOVA was used determine the effect of the treatments (Control, 65% SSP+ PAS, 75% SSP+ PAS and 85% SSP+ PAS) (Table 3) and days (Table 4) on the PSL markers and the interactions between the treatments and days (Table 5) on the PSL markers using 95% confidence interval.
 

 

 

 

There was statistically significant (p<0.05) difference between the swirling observed in the control and 65% SSP+ PAS added CPC group and that the swirling quality of 65% SSP+ PAS added CPC was well maintained till day 9 (Table 2) (Fig 1) The difference in mean pH between the control group and the test groups was significant and the plasma stored control CPC showed similar pH to that of 65% SSP+ PAS added CPC group (7.27±0.021) and (7.22±0.021) (Table 3). The mean pH of the control CPC on day 5 was 7.28±0.0384 which similar to that 9^th day of 65% SSP+ PAS added CPC (Table 5) (Fig 2).
 

 

 
The mean glucose in the control CPC (441±5.19 mg/dl) was significantly high (p<0.01) when compared to that of the SSP+ PAS added PC groups (Table 3). The depletion in glucose concentration was significant (p<0.01) in control CPC when compared to the PAS added test groups (Table 5) (Fig 3). Similarly, mean lactate concentrations evidenced a statistically significant difference (p<0.01) between the control group and the SSP+ PAS added CPC group (Table 3). The increase in the lactate concentrations and difference in the mean lactate concentrations through the days was high for the 65% SSP+ added PAS group (Table 4 and 5) (Fig 4)
 

 

 
The difference in the mean pO₂ and pCO₂ concentration between the control group, 65%, 75% and 85% SSP+ PAS added CPC group was significant (p<0.01) (Table 3) (Fig 5 and Fig 6). The difference between the mean bicarbonate concentration during storage in the control group was statistically significant (p<0.01) as was the difference in the mean bicarbonate concentration across the days of storage. (Table 3 and 4). The control CPC showed a rapid decline in the plasma bicarbonate concentrations when compared to the PAS added test groups (Table 5) (Fig 7).
       

 

 

 
The mean platelet concentration during storage in the control was significantly higher (p<0.01) when compared to the mean platelet concentration of the 65%, 75% and 85% SSP+ PAS added CPC groups and we observed that the platelet concentration remained fairly stable during storage for all the SSP+ PAS added CPC (Table 5) (Fig 8).
 

       
The MPV increased over time during storage across all groups and mean MPV for the control group, 65%, 75% and 85% SSP+ PAS added CPC evidenced a significant difference (p<0.01) across the treatment group (Table 3) The mean MPV at 9 days during storage was highest for 75% SSP+ PAS added CPC group while the lowest was observed in the control CPC group (Table 5) (Fig 9). The PDW also increased over time during storage across all groups and mean PDW for the control group, 65%, 75% and 85% SSP+ PAS added CPC group were not statistically significant (Table 3) interaction study between the additive concentration and the days for storage for PDW was unable to yield significant results (Table 5) (Fig 10).
 

 

       
All CPC samples under our study were subjected to bacterial culture examination and were negative for any bacterial growth.
       
It was observed that swirling was well maintained at moderate level in plasma stored control CPC up to day 6 of storage while it was maintained at moderate levels till day 9 in 65% SSP+ PAS added CPC group while the pH was within the range of 6.4-7.4 (Bertolini and Murphy, 1996). Swirling reduced drastically in plasma stored control CPC when compared to the 65% SSP+ PAS added group (Hlavac et al., 2017).
       
The pH dropped drastically during the period of storage in the plasma stored control CPC group which could be attributed to the increased utilization of glucose for platelet metabolism and subsequent increase in the lactate concentration leading to rapid decrease in the pH (Milford and Reade, 2016). The pH was maintained at a constant level and the decrease in the pH was not statistically significant for the CPC stored in 65% SSP+ PAS group which had a mean of 7.28 at day 9 of storage, similar to that of day 5 of the control group signifying that the quality of CPC was well maintained up to 9^th day of storage for the 65% SSP+ PAS added CPC group (Hoareau et al., 2014). Mean pH of 75% and 85% SSP+ PAS added CPC were above 7.4 which could be associated with loss of platelet viability (Tynngård, 2009).
       
The low glucose concentration in the PAS added CPC groups can be explained by the dilution of the plasma glucose by the addition of a PAS which contains very low amount of glucose and contains acetate instead as a fuel for platelet metabolism which leads to decreased consumption of glucose (Hlavac et al., 2017). The low mean lactate concentrations in the PAS added CPC groups can be attributed to the presence of potassium and magnesium in the additive solution which provides a buffering effect, influences the lactate production and preserves pH (Shanwell et al., 2003; Kiminkinen et al., 2016).
       
Unlike previous studies (Haines et al., 2020) the levels of pO₂ concentration were high when compared to the PAS added CPC groups which could be attributed to sampling error due to time duration between the collection of sample and time of analysis. Mean pO₂ increase during the period of storage indicate an increased anaerobic metabolism over aerobic even in the presence of oxygen leading to decreased pH and increased lactate production (Lasta et al., 2020). A decline in pCO₂ over time and subsequent increase in the pO₂ were indicative of a shift to anaerobic metabolism which can be associated with decrease in the pH that can be detrimental to the storage quality and viability of platelets in the control CPC (Tynngård, 2009; Stiegler et al., 2009).
       
The reduction in the bicarbonate concentration was steep in the plasma control CPC when compared to the PAS added CPC groups because of increased need for buffering the increased lactate production due to anaerobic glycolysis in the plasma stored control CPC (Hlavac et al., 2017).
       
The high platelet concentration in the plasma stored CPC group when compared to the PAS added CPC groups could be attributed to the increased dilution volume in the PAS added CPC groups (Haines et al., 2020). The decrease in the platelet concentration in the plasma stored control CPC showed a rapid decrease as compared to SSP+ PAS added CPC units because of the increase in platelet fragility and decreased viability during in plasma after day 5 of storage (Jain et al., 2015).
       
The increase in the MPV was conversely associated with pH of the concentrates (Singh et al., 2003). The mean MPV for the CPCs under the study was less than that reported by many authors (Bommer et al., 2008; Lasta et al., 2020) the reason for which require further investigations. Increase in PDW during storage could be attributed to an increase in the platelet size and increased platelet activation during storage as it is a sensitive indicator of platelet shape change (Matos et al., 2008, Schwartz et al., 2014; Souza et al., 2016).
       
All CPC samples were negative for any bacterial growth indicating the importance of proper aseptic collection.

The study of PSL markers in SSP+ added CPC concludes that 65% SSP+ PAS added CPC with 35% of plasma spill over was the ideal concentration to help maintain near ideal conditions for CPC storage with minimal deterioration in the quality and platelet viability. Further, ascertained by the retention of moderate swirling properties at day 9, maintenance of a constant pH within ideal limits when compared to day 5 CPC stored in plasma, flatter curve of increase in lactate and decreased bicarbonate consumption during storage.  The use of 65% PAS in CPCs was found to be advantageous by limiting the levels of lactate production, decline in pH and limiting bicarbonate consumption, thereby reducing the production of storage lesions. The increased duration of storage of CPCs will help increase the availability of the CPCs for treatment of severe thrombocytopenia and various bleeding emergencies in a veterinary emergency and critical care set up.

Tamil Nadu Veterinary and Animal Sciences University, Madhavaram Milk Colony Chennai, Tamil Nadu, India. Department of Veterinary Clinical Medicine, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu, India. Department of Clinics, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu, India. TANUVAS Animal Blood Bank, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu, India. Centralised Instrumentation Laboratory, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu, Indian Council of Medical Research (ICMR)-TANUVAS research (Scheme code 22293).

The authors declare that there was no conflict of interest.