Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

  • Print ISSN 0367-6722

  • Online ISSN 0976-0555

  • NAAS Rating 6.50

  • SJR 0.263

  • Impact Factor 0.5 (2023)

Frequency :
Monthly (January, February, March, April, May, June, July, August, September, October, November and December)
Indexing Services :
Science Citation Index Expanded, BIOSIS Preview, ISI Citation Index, Biological Abstracts, Scopus, AGRICOLA, Google Scholar, CrossRef, CAB Abstracting Journals, Chemical Abstracts, Indian Science Abstracts, EBSCO Indexing Services, Index Copernicus
Indian Journal of Animal Research, volume 57 issue 10 (october 2023) : 1296-1304

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

Paunas Kamlesh Joshi1, G.R. Baranidharan2,3,*, S. Kavitha1, A. Mangala Gowri4
1Department of Veterinary Clinical Medicine, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 007, Tamil Nadu, India.
2Department of Clinics, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 007, Tamil Nadu, India.
3TANUVAS Animal Blood Bank, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 007, Tamil Nadu, India.
4Centralised Instrumentation Laboratory, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 007, Tamil Nadu, India.
Cite article:- Joshi Kamlesh Paunas, Baranidharan G.R., Kavitha S., Gowri Mangala A. (2023). Evaluation of Platelet Storage Lesion (PSL) Markers in SSP+ Platelet Additive Solution Added Canine Platelet Concentrates (CPC) . Indian Journal of Animal Research. 57(10): 1296-1304. doi: 10.18805/IJAR.B-5150.

Background: To evaluate the Platelet Storage Lesion (PSL) markers and extended storage time for SSP+ added Canine Platelet Concentrates (CPC) beyond five days at 22°C.Fresh Canine CPCundergo certain metabolic and morphologic changes during storage. Estimation of PSL markers helps us better understand quality of the canine PC and to estimate the efficacy of SSP+ in increasing the life span of stored CPC.

Methods: CPC was prepared from 350 ml of whole blood, collected from 6 apparently healthy donor dogs by buffy coat methodin a quadruple bag closed system. CPC was divided into four aliquots of 15 ml each with one control and three test groups with SSP+ additive solution in different concentrations (65%, 75%, 85%). The In vitro PSL markers including swirling, pH, glucose, lactate, bicarbonate, pO2, pCO2, platelet concentration, MPV and PDWwere evaluated on day 1, 5, 9 and 13 of storage.

Result: The results were analysed by Two-way ANOVA wherein swirling, pH, glucose, lactate, bicarbonate, pO2, pCO2, platelet concentration, MPV showed significant difference (p<0.01) between the groups. Interaction between the additive concentrations and the days of storage revealed a pH on day 9 for 65% SSP+ to be similar to day 5 for Plasma stored CPC while swirling, lactate and bicarbonate were also better maintained for 65% SSP+ PAS added CPC till day 9.Addition of 65% SSP+ PAS to CPC evidenced increased shelf life up to 9 days at 22°C under agitation without significant deterioration in product quality.

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).
 

Table 1: Volume of additive solution for 65 per cent, 75 per cent and 85 per cent SSP+ PAS added PC groups respectively.


       
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.
 

Table 2: Effect of Mean±SE on platelet swirling (Kruskal Wallis test).


 

Table 3: Effect of additive concentration on Mean±SE of platelet storage lesion (PSL) markers (n=6).


 

Table 4: Effect of days of storage on mean±SE of platelet storage lesion (PSL) Markers (n=6).


 

Table 5: Mean±SE of platelet storage lesion (PSL) markers with interaction between additive concentration and days of storage (n=6).

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 9th day of 65% SSP+ PAS added CPC (Table 5) (Fig 2).
 

Fig 1: Day wise trend of mean swirling for control, 65%, 75% and 85% SSP+ PAS added PC.


 

Fig 2: Day wise trend of mean pH for control, 65%, 75% and 85% SSP+ PAS added PC.


 
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)
 

Fig 3: Day wise trend of mean glucose for control, 65%, 75% and 85% SSP+ PAS added PC.


 

Fig 4: Day wise trend of mean lactate for control, 65%, 75% and 85% SSP+ PAS added PC.


 
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).
       

Fig 5: Day wise trend of mean pO2 for Control, 65%, 75% and 85% SSP+ PAS added PC.


 

Fig 6: Day wise trend of mean pCO2 for Control, 65%, 75% and 85% SSP+ PAS added PC.


 

Fig 7: Day wise trend of mean bicarbonate for control, 65%, 75% and 85% SSP+ PAS added PC.


 
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).
 

Fig 8: Day wise trend of mean platelet concentration for control, 65%, 75% and 85% SSP+ PAS added PC.


       
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).
 

Fig 9: Day wise trend of mean MPV for control, 65%, 75% and 85% SSP+ PAS added PC.


 

Fig 10: Day wise trend of mean PDW for control, 65%, 75% and 85% SSP+ PAS added PC.


       
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 9th 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.

  1. Baranidharan, G.R. (2015). Clinicopathological and therapeutic evaluation of platelet disorders in dogs. Ph.D Thesis (Tamil Nadu Veterinary and Animal Sciences University). 

  2. Bertolini, F. and Murphy, S. (1996). A multicenter inspection of the swirling phenomenon in platelet concentrates prepared in routine practice. Biomedical Excellence for Safer Transfusion (BEST) Working Party of the International Society of Blood Transfusion. Transfusion. 36(2): 128-132.

  3. Bommer, N.X., Shaw, D.J., Milne, E.M. and Ridyard, A.E. (2008). Platelet distribution width and mean platelet volume in the interpretation of thrombocytopenia in dogs. J. Small Anim. Pract. 49(10): 518-524.

  4. Callan, M.B., Appleman, E.H and Sachais, B.S. (2009). Canine platelet transfusions. J. Vet. Emerg. Crit. Care. 19: 401-415.

  5. Haines, J.M., Hwang, J.K. and Wardrop, K.J. (2020). The effects of additive solutions on the development of storage lesions in stored canine platelet concentrates. J. Vet. Emerg. Crit. Care. 10.1111/vec.13031.

  6. Hlavac, N., Lasta, C.S., Dalmolin, M.L., Lacerda, L.A., de Korte, D., Marcondes, N.A. et al. (2017). In vitro properties of concentrated canine platelets stored in two additive solutions: A comparative study. BMC. Vet. Res. 13(1): 334. doi: 10.1186/s12917-017-1236-8.

  7. Hoareau, G.L., Jandrey, K.E., Burges, J., Bremer, D. and Tablin, F. (2014). Comparison of the platelet-rich plasma and buffy coat protocols for preparation of canine platele concentrates. Vet. Clin. Pathol. 43(4): 513-518.

  8. Hux, B.D., Martin, L.G. (2012). Platelet transfusions: Treatment options for hemorrhage secondary to thrombocytopenia. J. Vet. Emerg. Crit. Care. 22(1): 73-80.

  9. Jain, A., Marwaha, N., Sharma, R.R., Kaur, J., Thakur, M. and Dhawan, H.K. (2015). Serial changes in morphology and  biochemical markers in platelet preparations with storage. Asian. J. Transfus. Sci. 9(1): 41-47.

  10. Kiminkinen, L.K., Krusius, T. and Javela, K.M. (2016). Evaluation of soluble glycoprotein V as an in vitro quality marker for platelet concentrates: A correlation study between in vitro platelet quality markers and the effect of storage medium. Vox Sang. 111(2): 120-126.

  11. Lasta, C.S., Hlavac, N., Marcondes, N.A. et al. (2020). Quality control in veterinary blood banks: Evaluation of canine platelet concentrates stored for five days. BMC Vet. Res. 16: 25. doi: 10.1186/s12917-020-2254-5.

  12. Matos, J.F., Carvalho, M.G., Dusse, L.M.S., Ferreira, M.F.R. and Stubbert, R.V.B. (2008). The role of RDW, erythrocyte morphology and platelet parameters in the differentiation between microcytic and hypochromic anemias. Rev. Bras. Hematol. Hemoter. 30, 6. doi: 10.4081/hr.2018. 7605.

  13. Milford, E.M. and Reade, M.C. (2016). Comprehensive review of platelet storage methods for use in the treatment of active hemorrhage. Transfusion. 56(2): S140-S148.

  14. Schwartz, D., Sharkey, L., Armstrong, P.J., Knudson, C. and Kelley, J. (2014). Platelet volume and plateletcrit in dogs with presumed primary immune-mediated thrombocytopenia. J. Vet. Intern. Med. 28(5): 1575-1579.

  15. Shanwell, A., Falker, C. and Gulliksson, H. (2003). Storage of platelets in additive solutions: The effects of magnesium and potassium on the release of RANTES, beta- thromboglobulin, platelet factor 4 and interleukin-7, during storage. Vox Sang. 85(3): 206-212.

  16. Singh, H., Chaudhary, R. and Ray, V. (2003). Platelet indices as quality markers of platelet concentrates during storage. Clin. Lab. Haematol. 25(5): 307-310.

  17. Souza, A.M., Pereira, J.J., Campos, S.D.E., Torres-Filho, R.A., Xavier, M.S., Bacellar, D.T.L. et al. (2016). Platelet indices in dogs with thrombocytopenia and dogs with normal platelet counts. Arch. Med. Vet. 48: 277-281.

  18. Stiegler, G., Fischer, G., Ramanathan, G., Bencur, P., Weigel, G. and Mannhalter, C. (2009). P-selectin mRNA is maintained in platelet concentrates stored at 4 degrees C. Transfusion. 49(5): 921-927.

  19. Tynngård, N. (2009). Preparation, storage and quality control of platelet concentrates. Transfus. Apher. Sci. 41(2): 97-104.

Editorial Board

View all (0)