pathogen inactivation

1. PATHOGEN INACTIVATION
METHODS
Dr Atif Irfan Khan
Moderator: Dr Gopal Patidar

2. Outline
• Introduction
• Pathogen inactivation method for
• Plasma
• Platelets
• RBC
• Whole blood
• Prion elimination
• Potential limitations
• Summary

3. Introduction
• Transfusion transmissible infections poses significant threat despite
stringent screening processes
• With every unit of blood there is a 1% chance of transfusion
associated problems including transfusion transmitted diseases
Widman FK (ed) (1985) Technical manual. American Association of Blood Banks, Arlington, pp 325–344

4. Introduction
Assessment of NACO Supported blood banks A Preliminary Report 2016

5. Introduction
Level of safety in our blood donation centre
• Donor screening- through proper history and examination and
deferral
• Sample testing
• Chemiluminescence Immunoassay
• NAT
• Rapid test
• Quality control which detects blood product quality including culture
for organisms

6. Introduction
Routine procedures to minimise infection in blood processing
• Leukoreduction
Sources of TTI
• Infections not tested for routinely and missed during donor
screening
• Emerging infections
• Pathogens below detectable level by the serologic or nucleic acid
tests and donor being in window period of the infection
• Contamination during collection, storage and processing of blood

7. Introduction
• This warrants a method to completely eliminate the risk of infection
associated with blood transfusion
• Pathogen inactivation and pathogen reduction
• Terms have been used interchangeably but meaning differs
• PI should remove or inactivate all types of micro-organisms, without
inducing neo-antigens or reducing the function or lifespan of a
product
• When the method is unable to eliminate 100% of the pathogens it is
termed pathogen reduction

8. Introduction
Seghatchian and Putter described in 2013 various criteria of acceptability for
pathogen reduction treatments
1. Must be efficacious to eliminate a broad spectrum of pathogens and
prevent sepsis.
2. Should cause minimal damage to the blood transfusion product.
3. It must not compromise transfusion safety as assessed by in vivo assays
and clinical outcomes.
4. As an uncomplicated and cost-effective technology, it should be non-
toxic, maintain functional cell integrity for transfusion purposes, and pass
the stringent tests for bio-equivalency and bio-security.
5. It should satisfy the main criteria of availability: being accessible,
affordable, and safe, and demonstrate correct usage of the technology.

9. Introduction
Allain JP, Goodrich R. Pathogen reduction of whole blood: utility and feasibility. Transfusion Medicine. 2017 Oct 1.

10. Introduction- methods
• Chemicals: Physical disruption
• Solvent/detergent technology
• Photoactive compounds: Genomic disruption
• Psoralen derivatives (amotosalen)
• Riboflavin
• Methylene blue
• Chemicals: Short-term activation
• Genomic disruption S-303 (FRALE: Frangible Anchor Linker Extender)
• Direct radiation effect
• Genomic disruption- UVC

11. Introduction-principles
Photoactive compounds- basic principle of action

12. Chemical and Biological Mechanisms of Pathogen Reduction Technologies, Volume: 90, Issue: 5, Pages: 957-964, First published: 07 July 2014, DOI: (10.1111/php.12311)
Introduction-principles

13. Introduction-principles
Prowse CV. Component pathogen inactivation: a critical review. Vox sanguinis. 2013 Apr 1;104(3):183-99.

14. Pathogen inactivation(PI) of plasma products
Methylene blue(mb) method
• First pi method for plasma described in early 1990s
• Methylene blue (mb) is a phenothiazine positively pharged dye
• High affinity for negatively charged compounds.
• Activated by visible light, a photodynamic reaction generates
reactive oxygen species which target guanine and are responsible for
the nucleic acid damage
• Effective in damage of enveloped and some non-enveloped viruses.

15. PI of plasma products
Methylene blue(MB) method
• Some non-enveloped viruses, intracellular viruses, protozoa and
bacteria remain unaffected, further leukoreduction needed
• Thrombin generation capacity was reduced and clot formation
strength was unaffected
• Anaphylactic reactions reported in patients transfused with MB-Light
treated plasma

16. PI of plasma products
Solvent–detergent method
• Removes infectivity by disrupting the membranes of lipid enveloped
viruses, bacteria, protozoa and eukaryotes
• Comprise of 1% tri-(N-butyl)-phosphate (TNBP) and 1%
polyoxyethylene-p-t-octylphenol (Triton X-100)
• TNBP acts as an organic solvent which removes lipids from the
membranes, while Triton X-100 is a non-ionic detergent which
stabilizes TNBP and disrupts lipid bilayers for easier extraction of
lipids
• Treated for 1.5–4 hours at 30 °C.
• Cannot be used for PI of cellular blood components

17. PI of plasma products
Solvent–detergent method
• Non-lipid enveloped viruses like Parvovirus B19 and Hepatitis A virus
remain unaffected
• Some loss occurs of plasma proteins such as α2-antiplasmin and
Protein S.
• Another SD agent Octaplas FFP, introducing shortening of the
processing time, overcoming qualitative disadvantages of the past
SD method on the plasma product
• Some centers SD is not used in Protein S deficiency, IgA deficiency
• Carried out on pooled plasma

18. PI of plasma products
Psoralen light treatment
• Furocoumarins which include psoralens, are active compounds
isolated from plants
• Known as photo sensitizers since ancient times
• Amotosalen hydrochloric acid ( S-59) (INTERCEPT SYSTEM) has been
specially selected for PI of blood products
• it crosses plasma membranes efficiently
• demonstrates excellent protection against pathogens

19. PI of plasma products
Psoralen light treatment
• S-59 is activated by UVA which targets the helical region of single- or
double-stranded DNA or RNA, and intercalates instantly after
addition
• Upon exposure to UVA light S-59 binds covalently to pyrimidine with
continued exposure to UVA light a second bond is generated that
cross-links double-stranded structures, while single-stranded
structures are cross-linked in loops
• S-59 which is absorbed by haemoglobin, S-59 based PI cannot be
applied for red blood cells

20. PI of plasma products
• S-59 also binds to lipids and proteins
• Inactivate enveloped viruses, bacteria, protozoa and residual
leukocytes, but its effect on non-enveloped viruses is more variable.
• Retain pro-coagulant activity up to 80–90% of fresh plasma

21. PI of plasma products
Riboflavin light treatment (MIRASOL)
Acts by photodynamic mechanism
• Vitamin B2 act as a photosensitizer
• Mediates selective damage to nucleic acids upon exposure to
light(UVA and UVB, 285–365 nm), without binding to cells and
proteins
• RB associates with nucleic acids and mediates an oxygen-
independent electron transfer leading to nucleic acids disruption
primarily on guanine residues

22. PI of plasma products
Riboflavin light treatment

23. PI of plasma products
Riboflavin light treatment
• Results in in 20–30% reduction in fibrinogen, factor VIII and factor XI
activities
Marschner S, Goodrich R. Pathogen reduction technology treatment of platelets, plasma and whole blood using riboflavin and UV light. Transfusion Medicine and
Hemotherapy. 2011;38(1):8-18.

24. PI of platelets products
Amotosalen/UVA (INTERCEPT®; Cerus, Concord, CA)
• Mechanism of PI is same as described earlier
• Platelets are resuspended with plasma and platelet additive solution
to which amotosalen (150 μmol/L) is added.
• The mixture is then exposed to UVA light (3 J/cm2) for 4–6 minutes on
continuous agitation.
• Amotosalen and the photo by-products are removed by compound
adsorption device (CAD) for 4–16 hours at room temperature with
agitation.
• Platelets are transferred and stored in standard storage bags

25. PI of platelets products
• All these transfer steps lead to 10–15% of platelet loss
• EuroSPRITE trial suggested that Intercept-treated (buffy coat-derived)
platelet components stored for up to 5 days were comparable to
conventional platelets used for transfusion support of
thrombocytopenic patients
• The SPRINT trial was performed on a total of 645 patients with
thrombocytopenia patients transfused with Intercept-treated
platelets received more transfusions, had lower platelet CCI post-
transfusion

26. PI of platelets products
Riboflavin/UVB based pathogen reduction technology(MIRASOL®
PRT; Terumo BCT, USA)
• Mechanism of PI is same as described earlier
• Riboflavin at concentration of 50μM in an illumination/storage bag
with platelet concentrate unit.
• UV illumination (6.24 J/mL in plasma) in a controlled temperature and
agitation for 10 minutes
• 3% of platelets are lost due to transfer from bag to bag

27. PI of platelets products
• Photolysis of RB results in the formation of 2'-ketoriboflavin, 4'-
ketoriboflavin and formylmethylflavin.
• These are normal metabolites of RB and have been detected in
normal non-illuminated human blood.
• leads to increased platelet activation
• Shape change from discoid to sphere,
• Induces partial platelet aggregation
• Increased platelet glycolysis

28. PI of platelets products
Theraflex UVC-based pathogen reduction treatment (MacoPharma,
Mouvaux, France)
• UVC treatment uses exposure to short wavelength monochromatic
UVC light (254 nm)
• Interfere with DNA/RNA replication while preserving protein integrity
• Leads to intra-strand or inter-strand cyclobutane pyrimidine and/or
pyrimidine-pyrimidone dimers on nucleic acid, subsequently halting
replication

29. PI of platelets products
• The platelet units (in illumination bag) are placed in an UV illuminator
under continuous high-speed agitation
• Exposed to UVC light (0.2 J/ cm2 energy with double sided exposure)
for one minute.
• Platelets are then transferred to the storage bag
• Whole process takes 8 minutes
• Reduce the bacterial load by >4 log
• Inactivates many viruses
• May cause increased platelet metabolism

30. PI of platelets products
Storage lesion
• PSL is best defined as the sum of all the deleterious changes in
platelet structure and function that arise from the time the blood is
withdrawn from the donor to the time the platelets are transfused to
the recipient
• PI treatments may lead to an acceleration of storage lesions.
• Variety of proteins were affected (i.e., degraded, oxidized, or
phosphorylated), the number of altered proteins was low (relative to
the whole proteome)
• Majority of proteins remained intact.

31. PI of platelets products
Storage lesion
• PI has a moderate impact on platelet function, sometimes with
discordant results, illustrating the difficulties of assessing platelet
function in vitro.
• Storage lesions may be more pronounced, since increased P-selectin
expression and decreased agonist-induced aggregation was observed
• PI-treated platelets present a higher basic activation state
• Higher surface expression of GPIIb/IIIa; this could explain the faster
clearance, leading to lower recirculation rates, observed in some
clinical trials.
• However, hemostatic function appears to be preserved in PI treated
PCs compared to standard PCs

32. PI of platelets products
Prowse CV. Component pathogen inactivation: a critical review. Vox Sang 2013;104:183–99.

33. Chatterjee K, Zaman S, Chaurasia R, Singh S, Keil SD, Tewari S, Bisht A, Agarwal N, Rout D, Chand S, Saha K. Evaluation of
Mirasol pathogen reduction system by artificially contaminating platelet concentrates with Staphylococcus epidermidis: A
pilot study from India. Asian journal of transfusion science. 2016 Jul;10(2):127.

34. Inactivation of Zika virus in platelet components using amotosalen and
ultraviolet A illumination. Felicia Santa Maria et al
Method
Platelet components were spiked with
ZIKV, and ZIKV infectious titers and RNA
loads were measured by cell culture based
assays and real-time polymerase chain
reaction in spiked platelet components
before and after photochemical treatment
using amotosalen/ultraviolet A.
Outcome
The mean ZIKV infectivity titers and RNA
loads in platelet components before
inactivation were either 4.9 log10 plaque
forming units per milliliter, or 4.4
log10 50% tissue culture infective dose per
milliliter and 7.5 log10 genome equivalents
per milliliter, respectively. No infectivity
was detected immediately after
amotosalen/ultraviolet A treatment
Santa Maria F, Laughhunn A, Lanteri MC, Aubry M, Musso D, Stassinopoulos A. Inactivation of Zika virus in platelet components using amotosalen and ultraviolet A illumination. Transfusion. 2017 Aug 1;57(8):2016-25.

35. PI of RBC product
FRALE(Frangible anchor linker effectors) treatment
• The S-303 PI system (INTERCEPT RBC system, Cerus Corporation,
Concord, CA, USA)
• Acridine based S-303 is a positively charged alkylating compound
with 2 groups.
• The first group is an intercalating agent that locates to the helical
region of nucleic acids
• The second group is the effector molecule that allows covalent
modification of nucleic acid and degrades S-303 to S-300

36. PI of RBC product
FRALE(Frangible anchor linker effectors) treatment
• Glutathione is added to prevent protein damage caused by the
procedure due to acridine moieties on the RBC surface
• The first-generation S-303 procedure only marginally affected RBC
quality and function
• In the second-generation S-303(amustaline) system, GSH was
increased from 2 to 20 mmol/l in order to decrease the affinity of S-
303 for proteins and thus to avoid the formation of neoantigens on
the surface of erythrocytes

37. PI of Whole Blood
• Mirasol (Riboflavin/UVB) PRT is currently the single platform used to
reduce pathogens in whole blood, either prior to whole blood
transfusions
• Difficulties for PI is related to finding the optimal balance between
inactivation of pathogens with UV irradiation and damage it causes to
cells
• Use of amustaline (S-303) in WB appears more effective than the
riboflavin system for some infectious agents but does not permit the
preparation of acceptable blood components

38. PI of Whole Blood
• WB PI under going clinical trials
• WB units have been spiked with a number of pathogens, submitted to
WB PRT using the Riboflavin-UV illumination method (Mirasol) and
tested for reduction of infectivity with a range of methods
• Spiked viruses (HIV-1,HCV, Ebola virus); bacteria Gve+,Gve- and babesia;
parasites such asTrypanosoma, Leishmania and Plasmodium falciparum; and
nucleated blood cells present in the blood units
• Energy level applied to WB units over a period of 45–60 min with
intense stirring

39. In vitro studies examining the efficacy of WB PRT by mirasol
Allain JP, Goodrich R. Pathogen reduction of whole blood: utility and feasibility. Transfusion Medicine. 2017 Oct 1.

40. Effect of Plasmodium inactivation in whole blood on the
incidence of blood transfusion-transmitted malaria in endemic
regions: the African Investigation of the Mirasol System (AIMS)
randomised controlled trial
Jean-Pierre Allain et al.
• 1(4%) of 28 patients receiving blood
from parasitaemic donor developed
parasitaemia after mirasol treatment
• 8 (22%) of 37 patients patients
receiving blood from parasitaemic
donor without mirasol treatment

41. PI of prions
• Quinacrine and related heterocyclic compounds have been described
to exert anti-prion activity
• Continuous treatment with quinacrine might lead to the development
of drug-resistant or drug-induced mutated prions
• Octaplas has recently included a filter step (OctaplasLG), which as
shown by Heger et al. has achieved reproducible and high prion
infectivity binding capacity of ≥5.64 log(10) ID(50)/mL gel

42. Pathogen reduction methods in US and Europe in use and under trial

43. Other pathogen reduction technologies
Pasteurization of plasma products (wet heat treatment)
• This process has been in use since as early as 1948
• Inactivates viruses from albumin fractions
• This method is performed at 60 °C for 10 hours,
• 6-log reduction of the viral load
• 60–80% recovery of clotting factors.
• Non-enveloped viruses cannot be destroyed by this method
• Clotting factors are denature during this process unless additional
stabilizers are added

44. Other pathogen reduction technologies
Dry heat treatment
• Performed at 100 °C for 1 hour, leading to inactivation of lipid
enveloped and non-enveloped viruses
• there is a 90% recovery of clotting factors.
Acid-pH treatment
• (IVIG) and equine IgG preparations (antivenoms) are subjected to
pathogen inactivation by acid-pH treatment
• Incubated at pH 4, with or without pepsin, at temperatures from 30
°C to 37 °C, using protein content close to 50 g/L, and for more than
20–24 hours.
• Most of the enveloped viruses are destroyed

45. Other pathogen reduction technologies
Nanofiltration
• Involves size exclusion to remove virus particles and is also known as
viral filtration
• Reduces the viral load 5- to 6-log, including non-enveloped viruses
and preserves protein activity up to 90–95%
• Most plasma derived coagulation factors and immunoglobulins are
nano-filtered either after heat treatment, after pasteurization or after
SD method

46. limitations
• High cost of running the process; Mostly employed in developed
country for now
• Impact on the integrity of blood components and the toxicity of the
chemicals used in these systems
• May not completely eliminate all pathogens at the same time with the
same process
• No long term complications studied as of yet of PI application
• alkylating agents such as amatosalen may be carcinogenic in the long term
• No PI method approved as of yet for the most commonly requested
component; RCC

47. Summary
• Risk of TTI is cannot be overemphasised despite available
screening/detection techniques
• PI technology adds a layer of safety to the already available methods
of preventing TTI which is required
• Various methods inactivates pathogens in FFP & PC and maintain
functions of the blood component invivo to the set standards
• Efficient PI method for RCC & RBC still under study and development

48. References
1. Salunkhe V, van der Meer PF, de Korte D, Seghatchian J, Gutiérrez L. Development of blood transfusion
product pathogen reduction treatments: a review of methods, current applications and demands.
Transfusion and Apheresis Science. 2015 Feb 1;52(1):19-34.
2. Solheim BG. Pathogen reduction of blood components. Transfusion and apheresis science. 2008 Aug
1;39(1):75-82.
3. Prowse CV. Component pathogen inactivation: a critical review. Vox sanguinis. 2013 Apr 1;104(3):183-99.
4. Seltsam A. PATHOGEN INACTIVATION OF CELLULAR BLOOD PRODUCTS–AN ADDITIONAL SAFETY LAYER IN
TRANSFUSION MEDICINE. Frontiers in medicine. 2017;4:219.
5. Mundt JM, Rouse L, Van den Bossche J, Goodrich RP. Chemical and biological mechanisms of pathogen
reduction technologies. Photochemistry and photobiology. 2014 Sep 1;90(5):957-64.
6. Marschner S, Goodrich R. Pathogen reduction technology treatment of platelets, plasma and whole blood
using riboflavin and UV light. Transfusion Medicine and Hemotherapy. 2011;38(1):8-18.
7. Kaiser-Guignard J, Canellini G, Lion N, Abonnenc M, Osselaer JC, Tissot JD. The clinical and biological impact
of new pathogen inactivation technologies on platelet concentrates. Blood reviews. 2014 Nov 1;28(6):235-
41.
8. Allain JP, Goodrich R. Pathogen reduction of whole blood: utility and feasibility. Transfusion Medicine. 2017
Oct 1.