Introduction
Medicine is continuously exploring its possibilities in creating novel ways of providing cure for an array of diseases caused by pathogens. These pathogens are agents that cause diseases such as bacterium, virus or fungus. 2 The germ theory of disease of Pasteur, Koch and Loeffler defined the criteria for the causative relationship between agent and disease to be as follows agent is present in every case of a certain disease, agent is specific for that disease lastly, propagation of agent is possible through culture and its inoculation into nave host can cause the same disease. 3
In recent years, the utilization of blood transfusions in order to save lives has become well known. However, there have been concerns regarding its practice due to the spread of infections among subjects. Therefore, there arises the need for reducing the pathogens causing transfusion-transmitted infections through various methods primarily involving pathogen inactivation. 4
Pathogen inactivation is advantageous as it reduces possible occurrence of emerging infections, bacterial contamination or even the risk of transfusion-induced acute lung injury during cases wherein blood transfusion is necessary. Pathogen inactivation of blood components essentially offers the choice of utilizing one method of inactivating transfusion-transmitted infections, thus reducing the need for further testing. However, the present scientific advancements are still unable to suffice in inactivating all agents. Thereby, scientists ceaselessly give effort to characterizing various types of agents and devising inactivation techniques in correspondence to the aforementioned agents. 5
This dissertations main objectives are to identify of the pathogens subject for inactivation to characterize the types of pathogen inactivation techniques for blood components or products such as, red blood cells, leucocytes, platelets, plasma, fresh frozen plasma, factor concentrates, and cryoprecipitate and to provide the possible applications of these techniques in medicine.
Pathogens List
The pathogens which are targets of inactivation are, however, not limited to Hepatitis Viruses, HAV, HBV, HCV, HDV, HIVHBVHCV, Serological Vs DNA, HCV ELISA, HCV-RIBA, HCV-PCR, Epstein Barr (EBVHHV4) Virus, HHV-8, West Nile Virus, SARS, Cytomegalovirus (CMV) Neg blood for at risk patients, HTLV 1 and 2, miscellaneous viruses, protozoa Malaria, Syphilis ,Bacterial viruses e.g. Yersinia, Pseudomonas fluorescens, Retroviruses, Enteroviruses, Parvo virus B19, Prion Disease, and vCJD. These organisms are blood borne pathogenic microorganisms capable of causing diseases to humans and in order to inactivate these pathogens, the proceeding methods can be used.
Heating Treatment with the Use of Solvent
Biological products can be derived from human blood plasma and can be used in medical treatment. Inactivation of viral agents such as hepatitis B virus is essential for the success of these medical treatments. Examples of biological products that can be derived from the blood are plasma protein fractions. Plasma protein fractions are the usual subject of heat treatment with the use of solvent. These biological materials are heated to inactivate viruses. The process is done with the initial step of drying the plasma protein fractions such as albumin, antithrombin III, fibrinogen, factor VIII and prothrombin complex. Lyophilization is performed to dry the protein fractions and to make them in powdered form. An organic liquid plasma protein fraction suspension is formed by the adding inert organic liquid. The organic liquids that may be used are the following alkalines such as hexanes and heptanes, ketones, such as acetone and diethylketone, and perfluorochemicals, such as perfluorotripropylamine. Other organic liquids are viable for heat treatment as long as a suspension with the plasma protein fraction is formed. The suspension is, then, heated to a temperature of about 60o to 100 o C. The time by which the organic liquidplasma protein fraction suspension is subjected to such temperatures is dependent on how high the temperature is. After the entire process of heating, there will be the need to separate the organic liquid from the protein fractions through filtration. The protein powder shall, then, be air dried and further processed for patient use. This method is advantageous as it sterilizes protein materials whilst maintaining biological activity. It is also relatively easy to perform. However, due to the usage of heat for the process, there is a limitation to the types of blood components and products that can be subjected to it considering maintenance of biological activity.
Pasteurization
There is a known process of pathogen inactivation in dried plasma proteins and plasma protein fractions without essentially damaging the blood components biological activity. It is performed through heat treatment via pasteurization. The process is done through heat treatment of the suspensions of plasma protein fractions and dried plasma in a liquid organic heat-transfer agent. The heat-transfer agent can be symmetrical or mixed glycerol esters, which are liquid in pasteurization temperature. The heat-transfer agent can also be saturated or multiply saturated fatty acids possessing 4-22 carbon atoms. In addition, the combination of the preceding esters can also make up the inert heat-transfer agent with the maximum water content of the suspension to be 1 by weight and the temperature to be 50o-120 o C. Glycerol esters of saturated and unsaturated fatty acids are already known to be superb pasteurization mediums which can maintain biological activity whilst being easy to perform.
The pasteurization process is most of the time performed with the use of glycerol esters as heating agents without the need for pretreatment. On the contrary, the plasma proteins and plasma protein fractions such as antithrombin III, factor VIII and fibrinogen are used in dried form obtained, for example, through lyophilization, spray-drying, or other non-damaging drying methods. Aside from being safe and biological activity retaining, the pathogen inactivation using pasteurization has another advantage of being economical since the ingredients can be taken from natural sources. In addition, there is no toxicity side effect shown and the method is easy to perform. The disadvantage of pasteurization is the limitation in the number of biological products that can be subjected to it since the dried, biologically active form is necessary. 8
Solvent Detergent (SD) Treatment
The very first commercialized pathogen inactivation technique used in blood components is the solvent-detergent treatment of whole plasma. The method relies on its ability to damage lipid envelopes of viral agents through solvent-detergent treatment, which after processing is eventually removed. SD Treatment is well known worldwide and is used by many laboratories. 5
The emulsion provides a stable oil-in-water emulsion for inactivating viruses upon contact. The oil-in-water emulsion is composed of the oily discontinuous phase, which contains an organic phosphate-based solvent dispersed in aqueous continuous phase. The treatment is successful in viral inactivating in a way that if it adheres one of its components of the oil phase to the lipid envelope of a virus, then, the envelope structure is disrupted. This process is mostly similar with contacting di-or trialkylphosphate to a blood protein composition. The di-or trialkylphosphate used can be between 0.01mgml and 100mgml in amount. The treatment can be performed with or without wetting agents and the treatment of blood protein composition with trialkylphosphate is done at a temperature between -5oC and 70oC, preferably 0oC to 60oC. The time of treatment ranges from 1 minute to 24 hours. Considering atmospheric pressure, the normal is preferred although subatmospheric and superatmospheric are also allowed. Afterward, removal of the trialkylphosphate and other agents is performed. If ethers are present, the plasma is subjected to 4oC to 37oC with slight vacuum for additional efficiency in segregation. 9
The advantage of this technique is that emulsions are, essentially, stable, non-toxic, easy to perform and economical. There are, on the contrary, some disadvantages for this technique. The process is only applicable for lipid-enveloped viruses and is characterized to be cell damaging, thus, red blood cells and platelets are not viable for this method. 5 In order to assure the inactivation of non-enveloped viruses, there must be another viral inactivation such as pasteurization or dry heat. However, in recent years, there has been an increased knowledge on the inactivation of non-enveloped viruses using SD treatment. It has been discovered that the use of phenol andor formaldehyde in a previously SD treated product can also aid in the inactivation of non-enveloped viruses. The process of SD treatment stipulates using prequalified concentrations of formaldehyde and phenol with SD treatments. 10
Methylene Blue (MB) Treatment
Methylene blue (MB) is a photoactive phenothiazine dye used for pathogen inactivation in single units of plasma. Methylene blue has a high affinity for nucleic acids and surfaces of viruses. Viruses treated with MB when expose to UV light are readily inactivated. However, just like when using SD treatment, non-enveloped viruses are more difficult to penetrate. In order to enhance the MBUV treatment for intracellular viruses, there is a way to liberate them and expose them to the treatment. This is done through freezing and thawing plasma which disrupts the cell membranes of leukocytes. The process of MB treatment is performed with the initial step of adding methylene blue stock solution to individual thawed plasma resulting in 1M concentration. The next step is the subjection of the treated plasma to white light luminescence of 45000 lux or higher for an hour. 11
Residual intact white blood cells possessing viruses can be removed by a micropore filters. The disadvantage of Methylene blue treatment is that it can inactivate neither protozoa nor bacteria. In addition, plasma proteins subjected to it are moderately affected as well as fibrinogen and factor VIII activity, which are reduced by up to 30. Exposure to even minute amounts of residual MB and its photoderivatives have yet to be studied in larger scale. Fortunately, up to date there have been no adverse effects reported nevertheless, long-term studies of carcinogenicity and reproductive toxicity have not been conducted to prove this. 11
Amotosalen (Virus Inactivation of plasma)
The amotosalen treatment contemplates methods of inactivating pathogens in a biological composition which involves a compound selected from the group consisting of primaryamino-pyrone-linked psoralens and primaryamino-benzene-linked psoralens a photoactivating means for photoactivating said compounds and a biological composition suspected of being contaminated with a pathogen which must contain nucleic acid. The addition of the compound to the said biological composition is done and thus, photoactivating said compound, in order to inactivate said pathogen. The biological composition is a blood product thought of to be contaminated. The blood products utilized are platelets or plasma. After amotosalen treatment is performed, the blood product can be suitable for its intended use. 12
Aminomethyl-trimethyl psoralen is also known as amotosalen hydrochloride or S-59. It has been used to inactivate pathogens in plasma and platelets. The process of pathogen inactivation using amotosalen is performed through, initially, reducing volume of platelet concentrate to be suspended in 30 to 45 plasma and 70 to 55 platelet additive solution containing sodium chloride, acetate, citrate and phosphate. The amotosalen, 150molL, is added to the platelet and incubated for a period of 3 to 5 minutes. The subjection o the product to 3 Jcm2 of UV light for 3 minutes is, then, performed. In the previous step, agitation is necessary. After UV-A exposure, approximately 80 of the amotosalen were photodegraded to by-products. The residues of psoralen and by-products are removed by a compound absorption disc to avoid potential toxicity. The amotosalen-UV-A treated platelets are, then, placed in a bag containing the S-59 absorbent and incubated at room temperature. Agitation is done for 4 to 16 hours before transferring to the final platelet storage bag. The advantage of using amotosalen in pathogen inactivation is the process need for a lower dose of UV-A, thus, exposure is shorter and there is obtained corresponding avoidance of plasma damage. Also toxicity is very low if any. 11
Intercalation
S-303 (Helinx) is a molecule made for pathogen inactivation treatment of red blood cells. Helinx is an alkylating agent derived from quinacrine mustard characterized to be a frangible anchor linker effectors compounds. The Helinx technology is used in the INTERCEPT Blood System for Red Blood Cells. It uses the active compound S-303, a member of compounds known as FRALEs (Frangible Anchor Linker Effector). Frangible anchor linker effectors compounds possess an intercalator group that inserts into the helical region of DNA and RNA. Effector groups allow covalent linking of nucleic acids, and as well as a central frangible bond that organizes the degradation of the compound. S-303 can easily intercalate into the helical regions of the negatively charged nucleic acids and the process does not depend on light to pursue. A shift from a lower pH storage environment to the higher neutral pH of red blood cells causing hydrolysis activates intercalation. 11
Pathogen inactivation of components containing red blood cells has presented itself to be challenging. S-303 method is utilized on whole blood and red blood cells. Pathogen inactivation of an array of viruses, bacteria, and protozoa has been associated to S-303 which is characterized by its intercalating ability. The advantage of this technique is that there is no known toxicity. However, expertise is required in the performance of this pathogen inactivation technique. 11
Leukocyte Depletion
Leukocyte depletion is a common practice among blood banks as it was made available by Fleming almost a century ago. Its main purpose is to use filtration methods in removing leukocytes from the blood. The removal of leukocytes aims to hinder inflammatory response especially during heart surgeries and is said to decrease incidence of ventricular arrhythmias. In recent years, newly designed filtration methods are available which is enhanced by rapid flow yielding to an excellent leukocyte removal rate. These new filters remove 99.995 of the leukocytes from the blood. Unfortunately, during surgery the rate may be somewhat lower with a 96.8 removal of leukocytes for cardiopulmonary bypass perfusate. 13
Gamma Irradiation
Transfusion-associated graft-versus-host disease (TA-GVHD) is a type of disease that results from unsterile blood transfusion. The chances of obtaining transfusion-related disease is said to rely on the percentage of the contaminating lymphocytes in transfused blood, the susceptibility of the patient to the disease and the disparity between the patient and the donor. In order to attenuate this, Gamma irradiation is used in inactivating T lymphocytes while maintaining the biological function of other blood cells. Nevertheless, gamma irradiation shall not exceed 50 Gy. In spite of this, there is evidence that gamma irradiation results in lower post-transfusion red cell recovery if storage is prolonged. The same thing goes for granulocytes which, in order to prevent harmful effects, must be transfused as soon as possible after preparation. Hence, the use of Gamma irradiation presents its own risks for patient if performed improperly. In-depth knowledge regarding its practice is highly necessary. 14
Ultraviolet B Irradiation
Leukocytes in labile blood products are able to immunize recipients of donor HLA antigens. This can bring about induction of refactoriness. UV light was observed to damage HLA class II related surface structures on lymphocytes and monocytes. In addition UV light was also observed to remove proliferative responses to mitogens and alloantigens and as well as viability. Similar to leukocyte depletion, UV B irradiation is also able to inactivate contaminated leukocyteslymphocytes whilst maintaining biological function of platelets. The process involves treatment of normal PC with a UV B dose of 0.3 Jcm2 with a wavelength of 310 nm for the period of 22 minutes. This process is most applicable with patients who recently underwent bone marrow transplantation. UV B Irradiation is said to reduce, however not to completely eliminate, HLA alloimmunization and subsequent platelet refactoriness. 15
Quarantine of FFP
Fresh frozen plasma is a component for transfusion prepared either from entire blood or from solely plasma taken with the use of apheresis. FFP is frozen for a certain period of time with a temperature adequate in maintaining plasma proteins to be biological active. FFP contains 80-92 of plasma coming from donor and citrate coagulant which is dependent on the type of donation and hematocrit compatibility. 16Q-FFP has been developed to reduce the risk of HIV and HCV transmission. 17
Glycerolization
Glycerolization is performed to prolong the storage life of the blood components. Ideally, the glyceralization takes place within six days of collection of blood in CPD or CPDA-1. Freezing the blood components come after. Glycerol is a cryoprotectant that serves as a means for the maintenance of biological activity and, also, a limit provider for pathogen growth while being frozen. This is made possible by limiting the oxygen availability for the blood components. The method by which glyceralization is done is as follows glycerolized red cells shall have a final concentration of 40 WV of glycerol it can, then, be frozen at 80C over a period of 30 min using manual refrigeration afterwards, it can be preserved at -60 to -65C for 10 years. In the production of 40 WV of glycerol, the glycerolizing solution is composed of 6.2 M glycerol solution that consists 57 gm glycerol, 1.6 gm Na lactate, 0.03 gm KCl and a total of 25 mEq1 of monobasic and disodium phosphatesin order to create a 6.8 pH. Another concentration of glycerolized red cells is 20 WV of glycerol. It can be frozen at - 196C using liquid nitrogen for 2-3 minutes and be preserved in the gas phase of liquid nitrogen at -120C for 3 years after. 18
Nanofiltration
Nanofiltration, the most recent technique for pathogen inactivation, has been used in effectively inactivating infectious transmissible spongiform encephalopathy agents in animals also known to be agents of vCJD. 19 Since the 1990s, nanofiltration of plasma products has been available already to implement viral safety suitable for the pathogen inactivation of human immunodeficiency virus, hepatitis B and hepatitis C virus, and, even prions. Most recently, nanofiltration has been known to be a reliable viral reduction technique that can be used for all plasma products. Nanofiltration serves as a possible safeguard against novel infectious agents potentially entering the human plasma pool. The process can be done with the use of filters of defined pore size. It is characterized by filtering protein solution through membranes of a very small pore size, typically 15-40 nm. The sizes of the pores suit retaining viruses. The advantages of nanofiltration include flexibility, efficiency, predictability of removing more than 4 to 6 logs of an array of viruses, and non-denaturing for plasma proteins. Up to recent times, nanofiltration is the only known technology to remove enveloped and non-enveloped viruses while maintaining 90-95 of protein activity20
Inactine PEN110
The Inactine PEN110 technology is a pathogen reduction process that was known to be in Phase 3 clinical studies. The study investigated the ability to inactivate mycoplasmas in human blood. The manner of execution is done by incubating whole blood or RBC units inoculated with Mycoplasma arthritidis or M. pneumoniae with PEN110 for 24 hours at 23 degrees C. Buffer treatment was also used under the same conditions. Through this Inactine process more than 107 mycoplasma CFU mL in whole blood and RBCs can be inactivated. This suggests the susceptibility of mycoplasmas to pathogen reduction. 21
Riboflavin
Riboflavin is capable of inactivating pathogens in all the three major blood components namely red blood cells, platelets and fresh frozen plasma.18 Riboflavin is known o inactivate pathogens with the aid of light. The mode of action of riboflavin photo-inactivation is essentially via electron transfer reactions in nucleic acids. The photo-inactivation procedure of riboflavin plus light can be performed with the blood components treatment with riboflavin and UV light that may result in reduction in the activity levels of some pro-coagulant factors. 22
Conclusion
The preceding pathogen inactivation techniques have proved themselves worthy to be tried and tested as means of medical treatment in a wider scale. However, one should consider the various factors that these treatments are affecting. The economic value of the treatments is rather questionable. This is in consideration of the different processes needed in order execute the entirety of the treatment. Expensive tests that are done in order to obtain blood samples from donors, the treatment, itself, can have a high cost since there are necessary materials and skills for success, and the administration to the patient can pricy, as well. Health is also a concern. There are unexpected risks in these newly developed treatments as they may have side effect in the long term scale. There are advantages and disadvantages for these pathogen inactivation techniques. This is a matter for the people, the patients, to decide whether to avail of them or not.
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