1. Current Status of Pathogen Reduction Methods
Thank you. It is a pleasure and an honor to be invited to speak before you this evening.
I would like to review the potential benefits and drawbacks associated with implementation of pathogen inactivation technologies in order to help clarify exactly what may be gained by their introduction.
James P. AuBuchon, MD
President & Chief Executive Officer
Puget Sound Blood Center
Professor of Medicine and of Laboratory Medicine
University of Washington
Seattle, Washington

2. Transfusion safety is like an onion…H G P N C V I T S I N G E T R A N O D E C U I R V O L U N T E D
SAFETY
It has been said that transfusion safety is like an onion. Our current systems provide multiple layers of protection, beginning with the use of volunteer donors who are educated about transmission risks and extending to the use of sensitive testing techniques. Pathogen inactivation would potentially add another layer of safety against viruses and bacteria.

3. “Emerging” Pathogens
Ocean virus
Chikungunya virus; Dengue fever
Modified from: Morens DM et al. Nature 2004;430:242-9.

4. Pathogen Inactivation Methods
Currently Under Investigation or in Use
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

5. Methods: Available or Approaching
Plasma
Quarantined plasma
Solvent/detergent treatment
Methylene blue + light
Amotosalen + UV
Riboflavin + UV
UVC alone
Platelets
Amotosalen + UV
Riboflavin + UV
UVC alone
Red cells/Whole blood
S-303
Riboflavin + UV

6. PI Plasma: The Similarities
Reduction in procoagulant activity: 10-20%
Effect of implementation: Clinical utility
Rock G. Vox Sang 2011;100;

7. PI Plasma: The Differences
SD Plasma
↓Allergic reactions (1/50,000 units)
↓TRALI [0 ?]
NAT for non-enveloped viruses (HAV, B19)
Reduction in HMW vWF + ADAMTS-13 retention
Reduction in anticoagulant proteins  thrombosis [?]
MB Plasma
Slightly greater fibrinogen + F VIII reduction

8. Evaluation MB Plasma Clots by Thromboelastometry
Slower
MAXIMUM CLOT FIRMNESS
Strength OK
Less thrombin
THROMBIN GENERATION
CLOT FORMATION
Cardigan R et al. Transfusion 2009;49:

9. PI Plasma: The Differences
SD Plasma
↓Allergic reactions (1/50,000 units)
↓TRALI [0 ?]
NAT for non-enveloped viruses (HAV, B19)
Reduction in HMW vWF + ADAMTS-13 retention
Reduction in anticoagulant proteins  thrombosis [?]
MB Plasma
Slightly greater fibrinogen + F VIII reduction
Implementation  Increased use [?]
 Reduced TTP response
France: 11 severe allergic reactions (1 death)
UV ± Photosensitizer Plasma
UVC alone: ↓ F XI (no clinical trials)
Nubret K et al. Transfusion 2011;51:125-8.

10. PI Platelets: The Similarities
Some loss of platelets through process (small; manageable)
UV light  Identifiable platelet damage
Increased metabolic rate
Increased activation during storage
(↓mt DNA transcription)

11. Using UVC to Inactivate
Control Treated
Platelets 9.4± ±1.3 x 108/mL
HSR ± ±8%
pH ± ±0.05
Aggr: Collagen 62± ±7%
Glucose ± ±8 mg/dL
Lactate 12.5± ±1.0 mM
Illumination: 0.4J/cm2
Testing: Day 8
Walker WH et al. Vox Sang 2007;93(suppl 2):69.

12. In vitro Assessment of Functional Properties
Intercept 12
Mirasol 10 8
Control
Fibrinogen receptor expression, MFI 6 4 2
Control
TRAP-6 aggregation response,, %
Mirasol
Intercept
Picker SM et al. Transfusion 2009;49:

13. Treatment Effect: Metabolic Changes
Lactate, mM
Storage time, days
Picker SM et al. Vox Sang 2009;97:26-33.

14. PI Platelets: The Similarities
Some loss of platelets through process (small; manageable)
UV light  Identifiable platelet damage
Increased metabolic rate
Increased activation during storage
Reduced recovery
Reduced survival
15-25%

15. Clinical Trial: Amotosalen-Treated Platelets
The euroSPRITE Trial
Treated Control
Units transfused/patient p > 0.05
Count increment (109/L):
1h post-transfusion p < 0.02
24h post-transfusion p = 0.004
Corrected count increments:
1h post-transfusion , , p = 0.11
24h post-transfusion , p = 0.02
Because of the smaller doses received, the count increments were lower with treated platelets, not surprisingly. However, the corrected count increments were the same at 1hour after transfusion. The lower 24 hour CCI with treated platelets, however, may be reflective of their reduced survival. Understanding that an equal dose gave an equivalent count increment has focused attention on development of the system and on techniques that will reduce loss through the various manipulations.
Van Rhenen D et al. Blood 2000;96:819a.

16. Clinical Trial: Amotosalen-Treated Platelets
The SPRINT Trial
WHO Grade 2, 3 or 4 bleeding: No difference between groups
Platelet content of treated units: 7.5% less
Post-transfusion counts: 22-26% lower in treated group
Comparison by dose:
Equivalent effect from similar dose
The true clinical effect of Intercept platelets was addressed in the US clinical trial that used bleeding as it endpoint. Patients receiving treated platelets had no more bleeding episodes than those receiving control platelets.
Again, their increments were lower because the content of the units was lower.
As Intercept platelets are moving into routine use in Europe, however, these blood centers are not experiencing an increase in platelet transfusions. I’m sure Dr. Cazenave will comment on this in his presentation.
French/Belgian experience: No increase in usage
Loss: 8%
McCullough J et al. Blood 2001;98:450a.
Murphy S et al. Transfusion 2006;46:24-33.

17. Clinical Trial: Riboflavin-Treated Platelets
The MIRACLE Trial
n = 110
CCI1h: 31% decrease (primary outcome measure)
MAX
75%
MEAN
50%
25%
MIN
CCI1h
CCI24h
50,000
40,000
CCI
30,000
20,000
10,000
-10,000
Mirasol Control Mirasol Control
Transfusion 2010;50:

18. Clinical Trial: Riboflavin-Treated Platelets
The MIRACLE Trial
n = 110
CCI1h: 31% decrease (primary outcome measure)
CCI1h: 31% decrease (primary outcome measure)
No differences observed
Clinical bleeding assessment
Inter-transfusion interval
Transfusion 2010;50:

19. Pathogen-Inactivated Platelets in Routine Use
3 yr before  3 yr after adoption of INTERCEPT platelets
(Used in place of bacterial detection and gamma irradiation)
Before After
Patients
Transfusions
Transfusions/patient
Platelets collected/unit 6.6x x1011
Storage period 5d 7d
Outdating % 1.2%
Osselaer JC et al. Transfusion 2007;47:19A.

20. PI Platelets: The Similarities
Some loss of platelets through process (small; manageable)
UV light  Identifiable platelet damage
Increased metabolic rate
Increased activation during storage
Reduced recovery
Reduced survival
Interaction with leukocytes’ DNA 
Reduction in alloimmunization
Consideration of replacement of γ-irradiation

21. Prevention of Alloimmunization
MECHANISM INHIBITED BY PHOTINACTIVATED PI
MECHANISM NOT INHIBITED BY PHOTINACTIVATED PI
Marschner S et al. Transfusion 2010;50:

22. Prevention of Graft versus Host Disease
Adducts:
Amotosalen + UV 1/83 base pairs
Gamma irradiation 1/37,000 base pairs
Prevention of GvHD in murine model
Inhibition of APC function
Inhibition of cytokine production
R Dodd Vox Sang 2002;83(Suppl 1):
Osselaer JC et al. Blood 2007;110:849a.

23. PI Platelets: Concerns
SPRINT Trial (FDA)
Respiratory distress: 5 test vs. 0 control (n=671)
Independent, blinded review of all (148) pulmonary events
 No association with PI platelets
Corash L et al. Blood 2011;117:

24. Expected: ≥ 2 plt transfusions
PI Platelets: Concerns
HOVON Trial
Heme/Onc pts (n=295)
Expected: ≥ 2 plt transfusions
Plasma (n=99)
357 transfusion events
292 per protocol
PAS III (n=94)
381 transfusion events
278 per protocol
PR – PAS III (n=85)
391 transfusion events
257 per protocol
Early cessation:
Lower CCI1hr
Increased bleeding
Primary endpoint: CCI1hr
Secondary endpoints: CCI24hr, bleeding, transfusion needs
and intervals, reactions
Kerkoffs J-LH et al. BJH :

25. Expected: ≥ 2 plt transfusions
PI Platelets: Concerns
HOVON Trial
Heme/Onc pts (n=295)
Expected: ≥ 2 plt transfusions
Plasma (n=99)
357 transfusion events
292 per protocol
PAS III (n=94)
381 transfusion events
278 per protocol
PR – PAS III (n=85)
391 transfusion events
257 per protocol
SPONTANEOUSLY
REPORTED; UNBLINDED TRIAL
Primary endpoint: CCI1hr
Secondary endpoints: CCI24hr, bleeding, transfusion needs
and intervals, reactions
Kerkoffs J-LH et al. BJH :

26. APPROPRIATE TO COMBINE?
PI Platelets: Concerns
Maximum grade of bleeding (%)
Plasma PAS III PR – PAS III
Grade % 11% %
Grade % % %
Grade % %
APPROPRIATE TO COMBINE?
CLINICAL
SIGNIFICANCE?
Kerkoffs J-LH et al. BJH 2010.

27. 6-7d Storage of Amotosalen-Treated Platelets
Untreated PI Platelets
Patients
CCI1hr 9, ,163
CI1hr 21, ,400/μL
CI24hr 15, ,100
Interval d
HSCT: 67%
p < 0.05
Δ = 17% (<30%)
p < 0.05 NS
6d: 20% 7d: 80%
Lozano M et al. ISBT 2010.

28. Bacterial Reduction by Antimicrobial Peptides
Antimicrobial Peptides Studied
Thank you for the opportunity to present my views on how this country has succeeded – and fallen short – in addressing the problem of bacterial contamination of blood components, particularly platelets.
Before I begin my remarks on this subject, I would like to clarify for whom I am - and am not – speaking.
Mohan KVK et al. Transfusion 2010;50:

29. Bacterial Reduction by Antimicrobial Peptides
Mohan KVK et al. Transfusion 2010;50:

30. Methods: Available or Approaching
Plasma
Quarantined plasma
Solvent/detergent treatment
Methylene blue + light
Amotosalen + UV
Riboflavin + UV
Platelets
Amotosalen + UV
Riboflavin + UV
UV alone
Red cells
S-303
Riboflavin + UV

31. S-303 Mechanism of Action t1/2 = 25 min pH ACIDIC S-300-
NEUTRAL
 ACTIVATION
S-300-
NO NUCELIC ACID
INTERACTIONS
Similar to amotosalen but no UV activation required

32. Effect of S-303 on RBC Recovery and Survival 11 full-unit reinfusions
35d storage
Treated Control
24h Recovery % % p = 0.048
Survival d d p > 0.05
n = 29, paired
11 full-unit reinfusions
If we have effective methods for treating plasma and platelets, what about the most-frequently transfused component, red cells? Here, ultraviolet light cannot help us as it cannot penetrate a red cell unit. Cerus develop a compound, S-303, that could interact with and react and bind to nucleic acids without requiring ultraviolet activation. When the initial process was examined in radiolabeled recovery studies, it appeared that, again, there was some toll from the treatment with reduced recovery, although survival was maintained. However, unlike with platelets, a problem of immunologic reactivity was found in chronic transfusion studies.
Rios et al. Transfusion 2006;46:

33. Y Y Problems with RBC Pathogen Inactivation
NEOANTIGEN
As S-303 is reacting with pathogens and chemically altering them so that they cannot infect or multiply, it can also react with the surface of red cells, placing the acridine ring there to be recognized by natuuralyl occuring antibodies, which some people have, or to be recognized by the immune system. Some recipients of S-303-treated red cells were found to have demonstrable antibodies that would interact with the treated red cells. While these antibodies did not have any activity in a monocyte monolayer assay, there was concern that they might shorten the circulation of treated red cells.
It appeared that these antibodies were anti-acridine antibodies, blockable in in vitro testing through the addition of acridine. Thus although it was not clear that these antibodies had clinical significance, finding them indicated that an modified approach needed to be developed.
ACRIDINE Y Y
No Monocyte Mononuclear Assay activity.
North A et al. Vox Sang 2007; 93(suppl 1):167-8.

34. Y Problems with RBC Pathogen Inactivation
Modified S-303 Process: Glutathione: 2  20mM at neutral pH Y
ANTI-ACRIDINE
A modified process has been developed that uses a higher concentration of glutathione at a neutral ph. This technique does not result in modification of the surface of red cells that is detectable by potent anti-acridine antibodies. The modified process is now entering radiolabled autologous reinfusion trials.

35. S-303 Treatment and Immunogenicity Augmented Rabbit Model
Recently completed: Blinded crossover autologous reinfusion trial
UNTREATED RBCs
mS-303
TRMT
S-303
TRMT
RBC
Recovery
(log scale)
Rabbits immunized with S KLH
Time
North A et al. Vox Sang 2007; 93(suppl 1):168.

36. Riboflavin Treatment and Immunogenicity
Baboon Model 90
Unlabeled infusions: Days 0, 21, 42, 49
51Cr infusion: Day 56
Quinacrine mustard trmt
Riboflavin + UV
Control RBCs
RBC
Recovery
(%) 80 70 60 50 40 30
No Ab demonstrated 20 10
Ab demonstrated
Time (h)
Goodrich RP et al. Transfusion 2009;49:64-74.

37. Pathogen Reduction Methods
Current Status of
Pathogen Reduction Methods
Yes, PI works.
But can the system accommodate?

38. Impact of Conversion to PI Platelets
All Patients
Hematology Patients
Before PI
After PI
Platelet Transfusions Required
Osselaer JC et al. Transfusion 2009;49:

39. “If someone says it’s not about the money,
it’s about money!”
Intercept Platelet conversion experience - Strasbourg
Kit cost: 75€/apheresis unit
Personnel time: 3€
Costs avoided:
Bacterial detection: 30€
Per new test: 10€
For France: Cost neutral with apheresis proportion
85%  55%
Cazenave JP et al. Vox Sang 2007; 93(suppl 1):51-2.

40. Reduction of Economic Impact
APHERESIS
PLATELET
TREATED
APHERESIS
PLATELET
APHERESIS
PLATELET
TREATED
APHERESIS
PLATELET
TREATED
POOLED
PLTS
Plt
WHOLE
BLOOD
Plt
DOUBLE
POOL
Plt
TREATED
POOLED
PLTS

41. An opportunity to improve patient safety simplify blood banking.
Pathogen Inactivation Technologies
An opportunity to improve patient safety
and
simplify blood banking.
From my perspective, pathogen inactivation technologies offer a wonderful opportunity to improve patient safety while, at the same time, greatly simplifying our approaches, and thus I look forward to their implementation.
Thank you.