Chemical inactivation Photo-inactivation Solvent-detergent Plasma Photosensitizers Methylene Blue light treatment Psoralen Ultraviolet Light Treatment Riboflavin Light Treatment INTERCEPT System Mirasol system Platelet and plasma Pathogen Inactivation FRALE and azridine compounds

1. C
PATHOGEN INACTIVATION
TECHNOLOGIES FOR
CELLULAR COMPONENTS
Presented by- Dr. Shiny
Moderator- Dr. Anshul Gupta

2. Introduction
• Two interchangeable terms, pathogen inactivation (PI) and pathogen reduction
(PR) have been used for a proactive approach in reducing contaminating
pathogens in blood and blood components.
• But by definition, PI refers to the complete prevention of infectivity by a
pathogen, whereas PR refers to decreasing the amount of an infectious
pathogen

3. Properties of ideal PI agents
a. Should inactivate or remove all types of infectious agents.
b. Should not add neoantigens in the cellular blood components.
c. Should have no effect on the function or life span of a blood component.
d. Should not have any residual toxic effects.
e. Should not involve risk greater than any TTI risk to the patient.

4. Two basic mechanisms of PI methods
1. Chemical inactivation
2. Photo-inactivation
• The chemical inactivation- based on the addition of heterocyclic chemical
compounds, which interact with nucleic acid (both DNA and RNA).
• Photometric excitation with UV or visible light- leads to an excited
electronic state which reacts directly with the nucleic acid (photochemical
reaction) or secondary activation to form free oxygen radicals or reactive
oxygen in a photodynamic reaction (e.g., methylene blue).

5. Plasma Pathogen Inactivation
1. Solvent-detergent Plasma
2. Photosensitizers
a. Methylene Blue light treatment
b. Psoralen Ultraviolet Light Treatment
c. Riboflavin Light Treatment

6. 1. Solvent/Detergent Treatment
• SD treatment- most extensively used and best validated PI technology
• 50 to 60 million doses of SD-treated pooled fractionated plasma proteins have
been given without the reported transmission of enveloped viruses.
• However, the method does not inactivate nonenveloped viruses, and nano-
filtration or heat treatment has been introduced as a second PI step for many
fractionated plasma proteins.
• SD treatment was first licensed for clotting factors in 1985 and has been in use
for the treatment of plasma since 1991.

7. SD Treatment- Method
• Most frequently used : Combination of 1% tri-(N-butyl)-phosphate (TNBP) and 1%
polyoxyethylene-p-t-octylphenol (Triton X-100) for 4 hours at 30 to 31ºC.
• TNBP : acts as an organic solvent and removes lipids from the membranes; it is used
alone in some protocols for PI.
• Triton X-100 : a non-ionic detergent that disrupts lipid bilayers for easier extraction of
lipids; it also stabilizes TNBP. Inactivation is below virus detection levels (6 log) within
15 minutes
• SD plasma is prepared from a pool of plasma from many donors (630-1520) that is
additionally tested for non-enveloped viruses parvovirus B19 DNA and hepatitis E virus.

8. Advantages of SD Treatment
• A major advantage of SD treatment is that the inactivation reagents in general do
not affect proteins and protein activity in plasma.
• Pooling also dilutes and in some instances neutralizes antibodies and allergens,
and allows the production of ABO-independent universal Plasma
• Particularly important is that there have been no reports of transfusion-related acute
lung injury (TRALI) after transfusion of over 5 million units of SDPP in Europe
• Hemovigilance data indicate that febrile, allergic, or anaphylactic reactions are
reduced by 70% to 80% with SDPP

9. • An improved version of SDPP is ABO-universal plasma that eliminates the
need for ABO group-specific plasma. It is based on the principle that anti-A
and anti-B can be neutralized by soluble A and B antigens in plasma.
• Since 1996, a lyophilized ABO-universal SDPP, Bioplasma FDP (National
Bioproducts Institute, Pinetown, South Africa), has been prepared from low
titered plasma pools in South Africa

10. 2. Photosensitizers
2. a. Methylene Blue Light Treatment
• Methylene blue (MB) : a positively charged phenothiazine dye with high affinity for
negatively charged compounds, such as guanine, proteins, and some lipids.
• MB-treated plasma is the second most frequently used pathogen-inactivated FFP
• More than 4 million units have been used clinically in European countries.

11. • Advantage- MB light treatment is effective against enveloped and some
non enveloped viruses.
• Disadvantage- MB does not effectively permeate plasma membranes;
thus intracellular pathogens and leukocytes may not be fully inactivated.
• Therefore, the original protocol developed by Institute Springe introduced
a freeze-thaw step to destroy residual leukocytes.

12. METHOD
• Methylene blue (MB) is added to thawed FFP, followed by its activation using
visible light.
• MB intercalates with nucleic acids or binds to lipids, and following activation
results in nucleic acid strand breakage or lipid peroxidation, with subsequent
modification of surrounding membrane proteins.
• Plasma is refrozen after the removal of MB-using the filter. MB treated plasma
contains 15-20% less factor VIII and fibrinogen than untreated plasma.

13. 2.b. Psoralen Ultraviolet Light Treatment
• Furocoumarins, which include psoralens, are active compounds isolated from
plants- known as photosensitizers since ancient times.
• Amotosalen hydrochloric acid (also known as S-59) : is a synthetic psoralen
specially selected by Cerus (Concord, CA) for PI of blood components because
it crosses plasma membranes efficiently and demonstrates excellent protection
against pathogens

14. • This treatment is called INTERCEPT System – Amotosalen and UV light are
passed through the component which irreversibly damages the nucleic acids of
various viruses, bacteria and protozoans.
• Fibrinogen, Factor V, Factor VII, Factor VIII, and Factor X are reduced by
17% to 30%, while coagulation factor inhibitor activity is less affected
• The INTERCEPT Blood System device developed by Cerus uses S-59, UVA
illumination, and a Compound adsorption device (CAD) for the removal of
residual S-59 and metabolites

15. 2.c. Riboflavin Light Treatment
• In spite of phototoxic targeting of nucleic acids, vitamin B2 (riboflavin) is
generally recognized as safe by the US FDA.
• Navigant Biotechnologies (Lakewood, CO) has developed the Mirasol system for
riboflavin treatment of blood components.
• Mirasol has been adapted for PI of platelets and plasma, while treatment of red
cells and whole blood still is under development.
• Advantage- Extensive PI of selected viruses (including parvovirus), bacteria, and
protozoa in addition to leukocyte inactivation has been reported in platelet
concentrates

16. Platelet Pathogen Inactivation
US FDA has recently cleared Amotosalen and UVA light for pathogen
inactivation of apheresis platelets suspended in platelet additive solution.
1. Psoralen UV Light Treatment
2. Riboflavin-Treated Platelets
3. Thionine Light Treatment

17. 1. Psoralen UV Light Treatment
• The INTERCEPT Blood Systems device for PI of platelets is similar to the
one for plasma
• INTERCEPT treatment also inactivates mitochondrial DNA and inhibits
cytokine synthesis, while platelet metabolic functions seem retained.
• The system has been used for the production of more than 50,000 doses of
PI platelets for routine clinical use in 14 European countries, and is reported
to have replaced bacterial testing, gamma irradiation, and the use of
cytomegalovirus-negative platelets.

18. 2. Riboflavin-Treated Platelets
• similar to the one for plasma.
• It is less complex than the INTERCEPT system in that removal of reagents is
not performed
• After a Phase III study was completed in Europe in 2007, the Mirasol system
for platelet PI was granted CE marking for application under GMP conditions
in blood centers.

19. 3. Thionine Light Treatment
• The phenothiazine dye thionine and UV light combined with strong agitation is
effective in the PI of platelets, and development of a protocol is being pursued
by Blood Centers of the German Red Cross and MacoPharma.
• However, strong agitation could pose problems with irreversible activation of
platelets.

20. Red Cell Pathogen Inactivation
1. Alkylating agents
a. Frale compounds
b. Aziridine compounds
2. Photosensitizers
3. Riboflavin light treatment

21. 1. Alkylating Agents
1.a. FRALE Compounds
• Frangible anchor linker effector (FRALE) compounds.
• Common- S-303
• specifically inactivate DNA/RNA via covalent adducts to the nucleic acid
• METHOD- Treatment is performed at room temperature for 12 hours followed by
incubation with a CAD in the final storage container for 8 hours in order to remove
residual S-303/S-300
• After satisfactory results with in-vitro studies and genotoxicity tests, successful Phase
I and II trials were performed, followed by Phase III trials in cardiac surgery and
sickle cell/thalassemia patients.

22. 1.b. Aziridine Compounds-
• Common- PEN 110
• Compounds such as ethylenimine, have been used to inactivate vaccines for the
past 30 years.
• The method inactivates both enveloped and nonenveloped viruses, including
members of the Parvoviridae family, which is often difficult to accomplish.
• METHOD- The treatment is performed at room temperature for 6 to 24 hours,
followed by washing with unbuffered saline to levels below the limit of
detection of PEN110

23. 2. Photosensitizers
• PI of red cells with photosensitizers poses problems because of light absorption
by hemoglobin and the high number of red cells, which requires complex
technical adjustments to secure uniform distribution of light for the treatment
of RBC units
• Because of hemolysis following PI of red cells with these aromatic dyes, the
methods was abandoned.
• A new approach was opened by the discovery of flexible dyes that can act as
photosensitizers when rigidly bound to substrate but do not generate reactive
oxygen species when in solution

24. • A protocol for PI of red cells is designed with TP and DP-
Thiopyrylium (TP) is a flexible photosensitizer that intercalates with nucleic
acids and also binds to red cells.
Dipyramidole (DP) (vasodilatator, antioxidant, red cell band 3 ligand) acts
as a competitive inhibitor of TP binding to red cells
• This method inactivated model viruses including intracellular HIV 6 log, and
six bacterial species 5 log reduction
• Hemolysis increases within acceptable levels and most, but not all, red cell
properties are retained after 42 days of storage

25. 3. Riboflavin Light Treatment
• Pathogen activation of microorganisms and leukocytes is similar to Mirasol
treatment of platelets and plasma, while hemolysis and in-vitro red cell
properties are within acceptable limits
• The Mirasol system has now also been adapted for PI of whole blood, which
afterwards can be separated into red cells, platelets, and plasma

26. PRION ELIMINATION
• None of the previously mentioned PI technologies eliminate prions.
• However, a significant reduction in infectivity occurs during plasma
fractionation, resulting in relatively low risk for prion transmission with
fractionated plasma proteins
• Several companies are developing prion reduction filters for red cell
components and interesting studies are in progress with ligand
chromatography, which may reduce prion infectivity of plasma significantly

29. Advantages of PI
• This technique has shown great efficacy in reducing the infectivity of a number
of pathogens harmful to oncology patients including CMV, parvovirus B19,
and T. cruzi.
• Prevents major transfusion and hemolytic reactions like TRALI
• Prevention of transfusion associated Chickengunya, West Nile Virus,
Lymphocytic Choriomeningitis Virus
• Prevention of protozoan infections like leishmaniasis, babesiosis and
toxoplasmosis

30. Toxic effects of PI agents
• INTERCEPT treatment of plasma decreases fibrinogen, Factor V and Factor VIII
activity by approximately 17% to 30% and thrombin generation capacity is impaired in
plasma.
• In vitro testing of INTERCEPT treated platelets showed increased CD61 microparticle
formation, higher metabolic rate, accelerated metabolic changes and reduced agonist
induced aggregation responses, but no neo-antigenicity was observed.
• Riboflavin treated platelets also leads to some platelet inactivation as expressed by
increased metabolic activity and P-selectin expression.

31. SUMMARY
CE Mark already obtained and under wide use in hospitals-
• Solvent detergent treatment of FFP- most extensively used
• MB treatment of FFP
• Psoralen light treatment of platelets as well as FFP
• Riboflavin light treatment of whole blood, which subsequently could be separated
into plasma, platelets, and red cells.
Under trial-
• Alkylating agents treatment of red cells
• Riboflavin light treatment of FFP

32. References
• DGHS. Tranfusion Medicine Technical Manual 3rd edition
• Rossi’s Principles of Transfusion Medicine, Edition 4
• AABB Technical manual 18th edition