Deb Chen presented on modifying red cell concentrates through post-production treatments and their impact on red blood cell quality. Red blood cells undergo storage lesions during the standard 42-day storage period. Gamma irradiation is currently used to prevent transfusion-associated graft-versus-host disease but negatively impacts red blood cell quality by increasing hemolysis and potassium leakage. Emerging pathogen inactivation technologies aim to inactivate pathogens while preserving product quality but may also accelerate storage lesion changes in red blood cells. Further research is needed to balance blood product quality and safety.

1. Deb Chen
PhD Candidate | Devine Laboratory at Centre for Blood Research
2015-09-26 | BCSLS Congress, Kelowna
Modifying Red Cell Concentrates:
The Impact of Post-Production Treatments on
Red Blood Cell Quality

2. 2
Modifying Red Cell Concentrates: The Impact of Post-Production Treatments on RBC Quality
Outline
1. Red Cell Concentrates
• Production and Storage
• Red Blood Cell Storage Lesion
• Post-Production Treatments
2. Current Post-Production Treatment
• gamma-irradiation
3. Emerging Post-Production Treatment
• pathogen inactivation technology
4. RBC Hemolysis: Biomarker Discovery for Quality Assessment

3. Product Production and Storage
Red Cell Concentrates
Whole Blood (WB) Donation Differential Centrifugation Leukoreduction
Separation of Plasma &
Platelets from RBC
• Preserved in SAGM (saline-adenine-glucose-mannitol)
• Stored for a maximum of 42 days at 4 ± 2°C

4. Red Blood Cell Storage Lesion
• Biochemical and cellular changes in stored red cells:
• metabolic modulation (e.g. ATP depletion)
• morphological alterations
• hemolysis (i.e., rupturing of red blood cells)
Red Cell Concentrates
Image adopted from Hovav T, et al. Transfusion. 1999 Mar;39(3):277-81.
D1 D21 D35

5. Adverse Effects of RBC Transfusion Contrasted
With Other Risks
Red Cell Concentrates
Image from Carson KL, et al. Ann Intern Med. 2012;157:49-58.
HIV = Human Immunodeficiency Virus
HCV = Hepatitis C Virus
HBV = Hepatitis B Virus
TRALI = Transfusion-Related Acute Lung Injury
TACO = Transfusion-Associated Circulatory Overload
= per RBC unit transfused
= per person per year

6. Post-Production Treatments
• Gamma-irradiation
• RBC Washing
• Supernate Removal by Spinning
• Pathogen Inactivation
Red Cell Concentrates

7. Transfusion-Associated Graft Verses Host Disease
• Rare complication of blood transfusion
o Delayed presentation with fever, diarrhea, and characteristic rash
o Occurs when the recipient’s immune system is unable to recognize donor T cells as foreign;
whereas donor T-cells recognizes host cells as foreign and mount an immunological attack
• Extremely high morbidity and mortality rate
o with death occurring within 1 month in over 90% of cases
• No effective treatment
• Supportive care, corticosteroids, and cytotoxic agents
Current Post-Production Treatment: Gamma-Irradiation

8. Gamma-Irradiation Treatment
• Currently the only approved strategy to
prevent TA-GVHD
• How does it work?
• Ionizing radiation penetrates the nucleated cells
(e.g., T cells) and damages DNA or generates free
radicals that indirectly disrupts DNA integrity
• Prevents proliferation of donor T cells in recipients
Current Post-Production Treatment: Gamma-Irradiation

9. Impact on Red Cell Quality
• In Vitro Parameters:
• Increased hemolysis – adherence to regulatory guidelines
• Potassium leakage – risk of post-transfusion hyperkalemia
Current Post-Production Treatment: Gamma-Irradiation
• Current gamma-irradiation guidelines (US and Canada)
• Performed at any time during RCC storage
• Irradiated units may be stored until the end of allowable shelf life,
but no longer than 28 days after irradiation
May diminish post-transfusion recovery and lead to potential adverse outcomes

10. The effect of timing of γ-irradiation on hemolysis
Current Post-Production Treatment: Gamma-Irradiation
Weeksafterirradiation
4
0.48 ±
0.18
(n = 61)
0.54 ±
0.34
(n = 43)
3
0.39 ±
0.22
(n = 68)
0.43 ±
0.24
(n = 70)
0.66 ±
0.44
(n = 57)
2
0.23 ±
0.12
(n = 67)
0.25 ±
0.12
(n = 70)
0.39 ±
0.18
(n = 64)
0.47 ±
0.29
(n = 45)
1
0.10 ±
0.06
(n = 72)
0.13 ±
0.07
(n = 73)
0.24 ±
0.21
(n = 67)
0.26 ±
0.17
(n = 69)
0.28 ±
0.15
(n = 70)
2 3 4 5 6
Weeks before irradiation
A
n=896
Serrano, K., et al. Vox Sang. 2013. 106:379-381
1
4
7
10
13
16
19
22
25
28
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Days before irradiation
1.5-2
1-1.5
0.5-1
0-0.5
0 2 4 6
AABB
Council of Europe
Daysafterirradiation
B
British Council of Standard in Haematology

11. The effect of timing of γ-irradiation on potassium
Current Post-Production Treatment: Gamma-Irradiation
0
10
20
30
40
50
60
70
80
11 d12 d13 d14 d15 d16 d17 d21 d23 d24 d28 d30 d31 d32 d35 d37 d38 d39 d42 d
SupernatantPotassium(mmol/L)
Days Post Collection
40 d Irradiation
35 d Irradiation
28 d Irradiaiton
21 d Irradiation
14 d Irradiation
10 d Irradiation
7 d Irradiation
QMP 2012 5 d
QMP 2012 42 d
n=84
Serrano, K., et al. Vox Sang. 2013. 106:379-381

12. RBC Washing
• Removes about 99% of the non-cellular fluid in a unit of blood,
including plasma proteins, electrolytes, and antibodies.
• Saline washed RBCs are indicated for
1. massive transfusion
2. patients with a history of severe allergic reactions
3. neonates
Current Post-Production Treatment: Gamma-Irradiation

13. Emerging Challenges to Blood Product Safety
Emerging Post-Production Treatment: Pathogen Inactivation Technology
Image from USC Institute for Emerging Pathogens and Immune Diseases
Image from State of Queensland, Dept. Agriculture, Fisheries & Forestry
Image from The Endless City
Image from Cynthia Goldsmith, US CDC

14. Pathogen Inactivation (PI) Technology
Emerging Post-Production Treatment: Pathogen Inactivation Technology
Prevent Disease Transmission Preserve Product Quality
Proactive strategy to better ensure a safe supply of blood products
Think-Pair-Share

15. Current Pathogen Inactivation Systems
& Mechanism of Inactivation
• PI Systems for Platelet Concentrate
• Theraflex UVC
• Intercept Amotosalen + UVA
• Mirasol Riboflavin (Vitamin B2) + UV
• PI Systems for Plasma Concentrate
• Octaplas TNBP + Triton X100
• Theraflex Methylene Blue + Visible Light
• Intercept
• Mirasol
Emerging Post-Production Treatment: Pathogen Inactivation Technology
Image from MerckMillipore

16. Central Dogma of Molecular Biology
• Generally describes the direction of information
flux in molecular biology
• Damage at the level of DNA will subsequently
disrupts downstream processes
• Inhibit pathogen proliferation (DNA replication)
• Prevent disease transmission (pathogenic proteins or virulence
factors)
Emerging Post-Production Treatment: Pathogen Inactivation Technology
Image from Psiopticon

17. Application to Whole Blood?
Emerging Post-Production Treatment: Pathogen Inactivation Technology
 Component Effectiveness
Potential Adverse Reactions
 T-Associated GVHD
 T-Transmitted Disease
• Known pathogens
• Emerging pathogens
• Close infectivity “window”
 Production Lab Complexity
 Cost of PI Technology
 Routine Donor Testing
 Cost in Ind. Component Tx
Cost Benefit

18. Impact on Red Cell Quality (Riboflavin + UV)
Emerging Post-Production Treatment: Pathogen Inactivation Technology
0
0.2
0.4
0.6
0.8
1
1.2
0 1 2 3 4 5 6
PercentageHemolysis(%)
Weeks of Storage
untreated
treated
0
5
10
15
20
25
30
35
40
0 1 2 3 4 5 6
PotassiumLevel(mmol/L)
Weeks of Storage
untreated
treated
n=6
• RBC derived from PI treated whole blood showed accelerated
storage-related deterioration compared to those left untreated
Adopted from Schubert, P., et al. Transfusion, 2015. 55: 815–823.

19. Summary
Modifying Red Cell Concentrates: The Impact of Post-Production Treatments on RBC Quality
• RBC undergo biochemical and biophysical changes during storage
• Residual viable donor T-cells are the culprit for TA-GVHD
• Gamma-irradiation – current strategy
• Pathogen Inactivation Technology – emerging strategy
• Post-production treatments negatively impacts RBC product quality
• Percentage Hemolysis
• Extracellular Potassium Levels

20. Balancing Quality and Safety - Where Do I Fit In?
RBC Hemolysis: Biomarker Discovery for Quality Assessment
Product Quality Product Safety

21. Acknowledgements
Devine Lab
Dr. Dana Devine Christa Klein-Bosgoed
Dr. Peter Schubert Ahmad Arbaeen
Dr. Katherine Serrano Simi Karwal
Dr. Elena Levin Tony Fang
Dr. Zhong-ming Chen Branika Culibrk
Dr. Geraldine Walsh
Modifying Red Cell Concentrates: The Impact of Post-Production Treatments on RBC Quality
Financial Support
Canadian Blood Services Graduate Student Fellowship Program
Production Team
Volunteer Donors