Microbiological Aspects of Platelet Transfusion Bags

Abstract

A blood transfusion service is one of the salient departments of every health care service. Blood transfusion can save life of patient, but it can lead to serious health issues due to transmission of infections if not dealt in an appropriate manner. Therefore, researchers are aiming at making the blood transfusion safe. Bacterial contamination of platelet bags has been kept under research consideration. This is due to the fact that platelet bags provide ideal medium for the growth of bacteria. Storage of platelet bags at room temperature also contributes to this dilemma. Several methods have been incorporated for the prevention and removal of bacteria from platelet bags. This starts from good selection of donor. Then, several methods for detection of bacteria are used. It includes visual inspection of platelet bags before transfusion. Bacterial Cell wall detection, molecular techniques, endotoxin detection, automated blood culture systems, Scansystem, Dielectrophoresis, pH and Glucose, Flow cytometry and microscopic examination are also among the methods used for detection of bacteria in platelet bags. Pathogen reduction technologies are also being implemented in this field Amotosalen-HCl and Riboflavin are mainly used for platelet transfusion bags, while PEN-110 and S-303 are mainly used for RBC transfusion bags. Considering the importance of this area of research, we conducted a research on platelet bags. Our study included laboratory research and computational docking. In the laboratory, we selected 352 platelet bags on day 6 of storage. Under aseptic measures, 10ml sample was taken from platelet bag. Four ml sample was inoculated in aerobic culture bottle. Three ml sample was taken in sterile plastic tubes for pH, glucose and protein measurement by Multistixdipstick method. Three ml sample was taken in sterile red top tube for glucose and protein measurement by Roche kit using COBAS instrument. Bacteria detection was carried out by inoculation of platelets from platelet bag in aerobic bottle on Day 6th of blood component preparation as recommended by BACTEC. Bacteria were then further characterised by different gram stain, cultures and analytical profile index (API) strips. We found that one platelet bag showed growth of bacteria. Secondly, we also discovered that glucose determination with dipstick was an easy technique. Glucose content showed correlation with pH of platelet bags. Therefore, it gave an idea about the quality of platelet bag contents. It is a known fact that bacterial growth in platelet bags causes decrease in pH and glucose content. Therefore, we can suggest that dipstick technique can help in detection of bacterial growth. Sample can be taken from platelet bag tubing and can be tested for glucose content by dipstick during storage period. Further studies are needed to establish the relationship between glucose content in tubing and platelet bags during the storage period of platelet bags. In the laboratory, under aseptic measures, we also took 4 ml sample from platelet bags for cell counts. Cell counts were done using Sysmex analyzer XE-2100. Weights of platelet bag and platelet bag volume were also considered to calculate actual platelet count of platelet bags. We divided the platelet bags into 4 groups according to actual platelet count i.e. group 1-4 with platelet count of less than 3×1010 /unit, >3-5×1010 /unit, >5-7×1010 /unit and >7×1010 /unit, respectively. We found that as actual platelet count increased, pH and glucose content decreased. All of these were not good signs for the survival of platelets. Due to increase consumption of glucose by increased platelets, glucose content of platelet bags decreased. Similarly, increased platelets produced increase acids. All of this leads to decreased quality of platelets. On the other hand hemoglobin, RBC, WBC and PDW increased as the actual platelet count increased. This is also not good indicator for the patient due to infusion of unnecessary cells in the patient. Increased RBC can cause increase chances of hemolysis. While increased WBC count in platelet count can cause transfusion reaction related to WBC. Therefore, more platelets in platelet bag should be considered salient and larger studies should be conducted to confirm the finding of our study. Second part of our research included computational docking of different pathogen reducing agents against different receptors of platelets. Rationale for that was the appearance of different side effects after usage of these pathogen reducing agents in platelet bags. The most significant of them was bleeding episodes with Amotosalen Hydrochloride. Therefore, researchers are working on different aspects of pathogen reducing agents to find its different effects from different aspects. We wanted to see the effect of pathogen reducing agents on platelets. Computational docking is considered significant tool in the field of drug designing. Different tools are available for docking. We used Patchdock, Firedock and Autodock vina for the docking. Amotosalen Hydrochloride (PubchemCID:159598), S-303(Pubchem CID:6433104), PEN-110 (Pubchem CID: 9091413) and Riboflavin (Pubchem CID: 493570), SDF structures were downloaded through website http://pubchem.ncbi.nlm.nih.gov/compound/.This was docked against different receptors of platelets like Alpha2b beta3 integrin, Alpha5beta3 integrin, Alpha2beta1 integrin, P2Y1 and P2Y12 receptors. Then docking results were analyzed. We found that all four pathogen reducing agents showed enough docking to P2Y12 receptors. Therefore, we selected the Single Nucleotide Polymorphism (SNPs) of P2Y12 receptor and docked all four pathogen reducing compounds to thatSNPs. In conclusion, we observed that Amotosalen Hydrochloride had adequate docking with Alpha2b beta3 integrin, P2Y1 and P2Y12 receptor including the SNPs of P2Y12 receptor. While PEN 110, S-303 and Riboflavin showed enough docking with Alpha5beta3 integrin, P2Y1 and P2Y12 receptor. Several studies had shown the more bleeding episodes with Amotosalen Hydrochloride. If we concentrate, we can appreciate that only integrin or receptor, which is docked more for Amotosalen Hydrochloride than S-303, PEN-110 and Riboflavin, was Alpha2b beta3 integrin. Alpha2b beta3 integrin was not docked by S-303, PEN-110 and Riboflavin. Alpha2b beta3 integrin is most plentiful integrin on platelet’s membrane. It binds with fibrinogen on activation with ADP. ADP binds with P2Y1 and P2Y12 receptors. In our study, we have observed that Amotosalen Hydrochloride binds with all (Alpha2b beta3 integrin, P2Y1 and P2Y12 receptors), so Amotosalen Hydrochloride may disturb the function of all receptors, which results in more bleeding. On other side, PEN-110, Riboflavin and S-303 bound with only Alpha5beta3 integrin, P2Y1 and P2Y12 receptors. Alpha5beta3 integrin frequency on platelets is low and P2Y1 and P2Y12 receptors are mainly binding site for ADP. ADP binding helps in shape change mainly. It should also be noticed that PEN-110 and S-303 are largely used in RBC bags. So, they do not affect platelets on large scale in the peripheral transfusion because of dilution. Although, Riboflavin is also used in platelets bags but bleeding episodes are not being reported in comparison to Amotosalen Hydrochloride. This might be due to the fact that they affect only Alpha5beta3 integrin, P2Y1 and P2Y12 receptors. Further, wet studies might help to confirm this finding.