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Pathogen inactivation is a process of eliminating potentially infectious agents from components that threaten blood supply using various methods.Such approach is made to make blood transfusion safe and minimize contamination.the target of scuh methods is to damage the DNA and RNA of the pathogen disabling their replication in blood components whilst causing little or no damage to the cellular and plasma proteins .some of these methods are already in use or undergoing clinical trails.the methods vary accordingly with the type of blood component used.
Solvent detergent treatment was the first to be developed.It is mostly used for pathogen inactivation in plasma.It uses organic detergent and solvents to inactivate lipid enveloped viruses such as HIV,HCV,EBV and CMV.it does that by disrupting the lipid envelope of the virus thus preventing replication.this technique is ineffective against non lipid enveloped viruses.Solvent-detergent (SD) results when a virucidal detergent and solvent(solvent 1% tributyl phosphate and detergent 1% triton x100 for 4hrs at 30degress) are incorporated into processing of plasma from pools of approximately 12000 donar produces(Barbara J.Bryant & Harvey G.Klein(2007)pathogen inactivation:the definitive safeguard for blood supply.Archieves of pathology anf laboratory:may 2007,vol 131,No.5,pg719-733).These chemical needs to eb removes afterwards,Tri-butuyl phosphate be oil extraction and Triton by chromatography absorption.This causes disruption in cells therefore is not applicable for platelet and red cells.This method affects coagulation factors by affecting proteins suc as alpha 2 antiplasmin and anticoagualant protein S.According to articles a new SD method iis developed for mini pools upto 10-12 units maximum recovery of coagulation factors and avoiding large pooling of plasma.
Another effective method used for plasma is Nanofilteration.This method is used to filter viruses based on their size.it uses a sieving method being 15 to 40 nm in size effectively filtering smaller viruses.Unlike SD this method removes both enveloped and non enveloped viruses and even viruses smaller than the pores size.also effective in removing prion.the effectiveness of this method depends on temperature,pressure,flow rate of filteration and ratio of protein voulume to filter area.This method has been documented to show causing very little damage to the protein characteristics allowing recovery of 90-95% of protein activity.
Another useful method for plasma is methylene blue in combination with visible light.methylene blue attaches to the viral nucleic acid which is then exposed to UV light.This inactivates most enveloped virus in the .This method is not useful in inactivating non enveloped viruses,protozoa,bacteria and other intracellular viruses.the process of freezing and thawing damages the leucocytes which makes the virus more vulnerable to this treatment.MB method has said to affect the plasma proteins and also coagulation factors scuh as fibrinogen and factor VIII.According to most articles MB treated plasma cause a slow clot formation apart from that does not cause adverse transfusion reactions although long term studies on its toxicity and carcinogenicity has not been carried out.(AuBuchon 2011,McCullough 2007)
UVC light and gamma radiation is aso a useful method applied for plasma and platelet.this method is carried out in combination with photosensitizers.different wavelength lights are uses which causes changes in nucleic acid structure.Not does this only damages the pathogen but it also affects the proteins.despite this drawback it has been reported tha Uv treatment inactivates most of the viruses including HIV completely.As with other previously mentioned methods this method also affects the effectiveness of coagulation factors although no clinical trial for this has been performed.UV light tends to damage platelet,increase their metabolic rate and increase its activation during storage(AcBouch 2011)
Amotosalen method is used for both platelet and plasma products.It uses a synthetic porsalen compound,Amotosalen hydrochloride (S-59) .This compound when exposed to UVA light affects DNA and RNA structure by forming cross link bonds between them.formation of these bonds prevents replication and transcription.a wide variety of pathogengets inactivated using this method such as virueses(HIV,CMV,HPV),protozoa ad bacteria.It also modifies the DNA leucocytes causing inactivation of T lymphocytes.There this method has said to be useful in preventing transfusion reactions associated to leucocytes such as host vs graft disease and plate associated transfusion reaction(Canellini,Waldugel,Andregg,Tisset 2010) amotosalen allows less damage to platelet morphology as it uses UV light for a short period of time .Platelet concentrate concentrate is reduced and resuspened in about 70-75% platelet additives such as sodium chloride.acetate.citrate and phosphate followed by addition of amotosalen,incubation for 5 minutes and then exposure to UVA light.Byproducts are removed while platelet is transferred to another bag containing S-59 and further incubated for 4-16 hours with agiataion.(McCullough 2007,AcBouch 2010)As per journal articles this is the only method for which complete trails has been carried out showing very little difference to the normal platelet standard.treatment of plasma using this method is very similer to that of platelet,difference being no platelet additives are added.this can be kept for upto 1 year when frozen at -18 degress.the product is known as fresh frozen plasma.Plasma protein and coagulation factors activites is affected to a very minimal level.Cryoprecipitate can also be produced using this method although no clinical trails has been carried out.no toxicity levels has been reported using this method.
Riboflavin/UV light is another method employed in pathogen inactivation of platelet.riboflavin a naturally occurring compound found in foods. when exposed to UV light it causes oxidation of guanosine bases.this oxidation causes breakage of the DNA strands to an extent that is beyond repair.this change in structure prevents the DNA from replicating and transcribing.most of the pathogens gets inactivated including intracellular and cell associated HIV,bacteria and protozoa.platelet/plasma under goes Uv treatment in a temperature controlled environment in a bag containing Ribofalvin.As with the former method it doesnââ‚¬â„¢trequire removal of its byproduct as it is naturally occurring.In comparison the metabolic changes in Amolostalen is more pronounced compared to riboflavin, lowering the ATP levels due to mitochondrial based energy production .(AcBouch 2011)There is more glycolosis causing activation of platelet.no significant damage is caused to the coagulation factors although trails are still underway.If the storage exceeds 5 days platelet viability gets affected.This applies to both Psoralens and riboflavin.UV light method.(McCullough 2007)
Thionine is a phenolthiazine dye that can be used in combination of UV light to inactivate pathogens in platelets.Advantage of this method being it also uses UVB light which increases the effectivenss of pathogen inactivation although clinical trails are still underway.as with other methods there is a little change in plate morphology and its chemical composition.
The most challenging of all is pathogen inactivation in red blood cells due to increased chances of hemolysis and decrease survival of RBC.Techologies for pathogen reduction of red blood cells are still under development such methods include S-303,PEN 110,Riboflavin/UV light,Dimethylene blue and flexible phtosenzitizer dyes.
S-303 (FRALE- fragible anchor linker extender) is an alkalyting agent that targets the nucleic acids of viruses, parasites,bacteria and protozoa without depending on light for activation.S-303 is positively chared which attaches to the negative nucleic acid of the pathogen causing hydrolysis preventing replication.RBC functions appears to be normal using this method whislt in vivo functions are more than 75%.despite this as with all methods there is a drawback of this method too.there is formation of antibodies against the RBC treated with S-303 although this doesnââ‚¬â„¢t create any clinical problems.
A similar method PEN 110 which is an INACTINE compound is shown to inactivate a variety of viruses,protozoa and even mycoplasma.Inactine a positively charged ethlyeneamine binds to negatively charged phosphate.the activation caused by this bonding causes the nucleic acid strands to break preventing replication.leucocytes are removed before addition of PEN 110 followed by incubation at room temperature.the cells are then washed and preservative solution is added.This can be stored for 42 days.As with S-303 there is no formation of antibodies against the treated RBC.there is minimum hemolysis and red cell antigens are not affected to a great extent.According to journal articles not enough clinical trails has been run to rule out its immunoreactivity and immunogenicity.
Riboflavin/UV light is similar to that used in platelet pathogen inactivation.Varying wavelength of light(280-350 nm) is used which not only inacitvtes pathogens but also leucocytes.RBC recovery is not shorted nor is there formation on of antibodies.This technique ais still undergoing trail.(AcBouch 2011 , McCullough 2007)
Dimethylmethlene blue is a proactive dye that has a high affinity ot nucleic acid.when it undergoes visible light it causes inactivation of DNA and RNA in RBC.compared to other methods this method affects RBC to a great extent causing hemolysis.Rbc is washed with normal saline and resuspended in an additive solution which is then incubated with dimethymethlene blue before illumination.this affects RBC intregrity although no clinical trials has been reported for this method.
The last method t mentioned is the usage of photosenziter dyes such as Thioprylium(TP) and Thiozole orange.these dyes attach and penetrate viral membrane affecting their capsids and nucleic acids under a certain wavelength of light.. not only does this reduce photochemical damage to RBC but tends to inactivate numerous intracellular viruses(HIV),many gram positive and gram negative bacteria.A drawback for using this method is that it damages RBC membrane during phototreatment.it causes potassium leakage from RBC to a great extent.According to articles large scale studies has not been carried out using this method.