1. Additive solutions for RBC storage: AS-1, AS-3, AS-5 & AS-7
Stephanie N. David, MD

2. Outline History & introduction of additive solutions
Comparing additive solutions used today
AS-7
Additive solution use in neonates
Current VUMC practice of selecting different AS for pediatric vs. adults

3. Blood Transfus 2012; 10 Suppl 2: s7-11
History
1970s: Saline-adenine-glucose (SAG)
1981: addition of mannitol  SAGM
No new RBC additive solutions have been licensed for use for over 20 years…until now!
First AS was SAG developed in the 1970s by European researchers
In 1981, the same group added mannitol to protect the RBC membrane and decrease hemolysispermitted up to 6 weeks refigerated storage of RBCs  this product is called SAGM. To this day, it is the most widely used RBC AS but not in US bc its not licenseed by FDA
The AS we currently use are variations of SAG & SAGM
Blood Transfus 2012; 10 Suppl 2: s7-11

4. Importance of Additive Solutions
During storage, RBCs undergo complex and progressive accumulation of changes—RBC storage lesion
These complex changes include…

5. RBC storage lesions Morphological changes
Slowed metabolism with decrease in concentration of ATP
Acidosis with decrease in concentration of 2,3-diphosphoglycerate (2,3-DPG)
increased affinity of hemoglobin for oxygen decreased capacity of rbcs to release oxygen
Morphological changes
Vary from smooth discoid shape to various membrane protrusions or spicula (echinocyte) to spheroid-shaped (spheroechinocyte)
Reversibility depends on storage duration
- Slowed metabolism with decrease in concentration of ATP
- Acidosis with decrease in concentration of 2,3-diphosphoglycerate (2,3-DPG)
increased affinity of hemoglobin for oxygen decreased capacity of rbcs to release oxygen
Blood Transfus 2010; 8:82-8.

6. RBC storage lesions continued
Loss of function of cation pumps
Oxidative damage with changes to structure of RBCs
Apoptotic changes
Loss of parts of the membranes through vesiculation
Loss of function of cation pumps
loss of intracellular potassium and accumulation of sodium within cytoplasm
*use in neonates
Oxidative damage with changes to structure of RBCs
Formation of hydroxyl radicals that can damage protein and lipids
Apoptotic changes
Loss of parts of the membranes through vesiculation
Cell activates vesiculation to eliminate proteins and lipids that have been damaged by oxidative stress
Blood Transfus 2010; 8:82-8.

7. Potential consequences of RBC storage lesions
Compromise safety and efficacy of RBCs
Reduce capacity to carry and release O2
Promote release of potentially toxic intermediates
Free hemoglobin can act as source of reactive oxygen species
Increased capacity of RBCs to adhere to endothelium
Enhanced thrombogenic or pro-inflammatory potential
Compromise safety and efficacy of RBCs
Reduce capacity to carry and release O2
Promote release of potentially toxic intermediates
Free hemoglobin can act as source of reactive oxygen species
Increased capacity of RBCs to adhere to endothelium
Enhanced thrombogenic or pro-inflammatory potential
The clinical consequences of storage lesions, however, remain a matter of persistent controversy
studies have reported heterogeneous and conflicting findings about the effect of blood storage duration on morbidity and/or mortality in trauma, cardiac surgery, and intensive care unit patients.
“Effects of Red-Cell Storage Duration on Patients Undergoing Cardiac Surgery” by Dr. Young (N Engl J Med 2015; 372: )
did not find that the transfusion of red cells stored for 10 days or less was superior to the transfusion of red cells stored for 21 days or more among patients 12 years of age or older who were undergoing complex cardiac surgery.
Blood Transfus 2010; 8:82-8.

8. Complex inter-relationships
RBC biochemistry
RBC cytoskeletal structure
RBC membrane proteins
difficult to predict how RBCs respond to different storage conditions
In summary—there is a Complex inter-relationship between RBC biochemistry, cytoskeletal structure, and membrane properties difficult to predict how RBCs respond to different storage conditions
Blood Transfus 2012: 10 Suppl 2: s7-11

9. Standard requirements for patenting new AS in USA
Level of hemolysis: below threshold of 0.8% at the end of the storage period
Survival rate of transfused cells: >75% at 24 hours after transfusion
Level of hemolysis: below threshold of 0.8% at the end of the storage period
Survival rate of transfused cells: >75% at 24 hours after transfusion
Assessed by measuring half-life of radioactively labeled rbcs
Very general parameters
Can easily be affected by biological variability between donors
Blood Transfus 2010; 8:82-8.

10. Anticoagulant/Preservative solutions
Acid Citrate Dextrose (ACD): 21 day storage
Citrate-phosphate-dextrose (CPD) & Citrate-phosphate-dextrose-dextrose (CP2D): 21 day storage
Citrate-phosphate-dextrose-adenine (CPDA-1): 35 day storage
Similar to CPD but mg adenine

11. Additive Solutions shelf life: 42 days AS-1: Adsol AS-3: Nutricel
AS-5: Optisol
AS-7: SOLX
Increases shelf life of RBCs to 42 days
AS-1,3 and 5 have all been in use in the USA and as far as I know, perform equivalently

12. Preparation of RBCs with AS
Blood collected in CPD or CP2D
Spun
Mixed with 110 mL AS for 500 mL collections (100mL for 450 mL collections)  product with HCT 55-65%
From UCSF transfusion service lecture

13. Here we have a table showing the contents of the anticoagulant-preservative solutions and ASs from the AABB website
Ill just point out that for the anticoag-preservative solutions, as I said earlier, ACD-A , CPD and CP2D all don’t have adenine, and all provide a shelf life of 21 days, while CPDA-1 does have adenine which increases its shelf life to 35 d
As for the Additive solutions, All have varying amt of dextrose and adeneine
AS-1,3,5 all have NaCl while AS-7 does not
AS-3 is only one with monobasic sodium phosphate
AS-7 is the only one with dibasic sodium phosphate
AS-3 is only one without mannitol
AS-7 is the only one w/ sodium bicarb and only one w/o sodium chlordie
AS-3 is the only one with sodium citrate and citric acid
CIRCULAR OF INFORMATION FOR THE USE OF HUMAN BLOOD AND BLOOD COMPONENTS. 2013

14. AS-7 (SOLX) Alkaline Previously called EAS-81 (Experimental AS-81)
Designed to improve RBC metabolism during storage by increasing the range and capacity of pH buffering by adding phosphate and bicarbonate
April 19, First AS approved by FDA in US in >20 years!
Approved for RBC storage at 1-6C for up to 42 days after collection
Result of collaborative effort between the US Army, The University of Cincinnnati and Hemerus Medical—a Minnesota based company
Alkaline
Previously called EAS-81 (Experimental AS-81)
Designed to improve RBC metabolism during storage by increasing the range and capacity of pH buffering
April 19, First AS approved by FDA in US in 25 years!
Approved for RBC storage at 1-6C for up to 42 days after collection
Final composition of AS-7 is reached after addition of acidic glucose component—which is part of the collection set but only added during the RBC processing phase
Transfusion March; 55:

15. In March 2015, so now 2 years after FDA approval, this study showed how AS-7, using AS-1 as a control, reduced the red blood cell storage lesion
Transfusion March; 55:

16. Study design & Methods
Storage quality measured in prospective, randomized and three-center trial
Subjects randomly assigned to have whole blood collected and processed into RBC and plasma components stored in one of 3 ways:
Control: processed within 8 hours and stored in AS-1 at 1-6°C for 42 days (n=60)
Test: processed within 2 hours and stored in AS-7 at 1-6°C for up to 56 days (n=60)
Test: Processed within 8 hours and stored in AS-7 at 1-6°C for up to 56 days (n=60)
Transfusion March; 55:

17. As I said earlier, this study used AS-1 as the control
As I said earlier, this study used AS-1 as the control. Again we can see here that compared to AS-1, AS-7 does NOT have NaCl but does have bicarb, and phosphate, and is alkaline---otherwise fairly similar.
Transfusion March; 55:

18. ATP
AS-1
Day 42
AS-7
Day 56
Intracellular RBC ATP concentration μmol/g Hb
4.4 ± 0.7  3.6 ± 0.8
4.3 ± 0.7  3.9 ± 0.7
4.3 ± 0.7 3.1 ± 0.8
Both day 42 & 56 AS-7—within the levels considered sufficient to maintain RBC viability
ATP recoveries (assessed as % of ATP at prestorage level) were significantly higher in AS-7 than AS-1
AS-1
<8 hr storage
AS-7
<2 hr storage
6-8 hr storage
ATP recovery (%)
At day 42
82.5 ± 14.6
93.7 ± 17.9
89.5 ± 13.7
Transfusion March; 55:

19. Morphology index
AS-1
<8 hr storage
AS-7
<2 hr storage
6-8 hr storage
Morphology index
Day 42
69 ± 8
81 ± 8
80 ± 6.6
Day 56
79 ± 7
77 ± 7
Shed microvesicle protein (mg /dL)
28.9 ± 18.2
At day 42
16.8 ± 11.3
At day 56
By day 56, AS-7 maintained 10% higher morphology indices than day 42 AS-1
Day 56 AS-7 RBCs contained 40% less shed microvesicle protein than day 42 AS-1 RBCs
Since Storage results in reduced integrity of RBC membrane  loss of biconcave shape
Morphology index= scale up to 100 for a perfect biconcave RBC
Transfusion March; 55:

20. Hemolysis in AS-1 units is significantly higher than AS-7 at 42 days
0.39 ± 0.21
0.29 ± 0.11
0.42 ± 0.17
Hemolysis in AS-1 units is significantly higher than AS-7 at 42 days
AS-7
Day 42
Day 56
Recovery (%)
88 ± 5
82 ± 3
All units stored for 42 days had recoveries > 75%
RBCs stored in AS-7 for 42 days had higher 24-hr in vivo recovery than historic controls (82 +/- 7%) in current AS
AS-7 at 56 days maintains 24-hr recoveries over the minimal FDA requirements
Hemolysis in AS-1 units is significantly higher than AS-7 at 42 days
24 hr recovery of RBCs as an assessment of storage lesion reslting in early in vivo death
**current AS allow RBCs to be stored for up to 42 days with a mean in vivo recovery at the end of storage of 82 +/- 7%, with 12% of samples showing less than 75% recovery, and hemolysis of 0.4%-0.5%
Transfusion March; 55:

21. Summary of AS-1 vs AS-7 Improved biochemical status
Decreased vesicle formation
Reduced hemolysis
Increased in vivo recovery at conventional and prolonged period of storage
Transfusion March; 55:

22. Red cell transfusions in neonates
Traditionally, used fresh CPDA-1 units (<7 days)
Initially concern for using AS due to mannitol (AS-1, AS-5) & adenine additives
Also concern about hyperkalemia, acidosis and decreased function of older red cells
Historically, use fresh CPDA-1 units (<7 days) because of a concern for additive solution toxicity in ASs
Initially concern for using AS due to mannitol & adenine
Mannitol, potentially causing renal damage since it is an osmotic diuretic
Adenine, potentially causing hepatic damage since its metabolized by the liver
Also concern about hyperkalemia, acidosis and decreased function of older red cells in these additive solutions with longer shelf lives
Transfusion 1996;36:
Transfusion and Apheresis Science 24 (2001):

23. AS use in neonates
Strauss RG, Burmeister LF, Johnson K, et al. AS-1 red cells for neonatal transfusions: a randomized trial assessing donor exposure and safety. Transfusion. 1996;36(10):
Red cells stored in AS-1 <42 days from single donor vs red cells stored in CPDA-1 stored <7 days
No significant differences in:
Pre-transfusion and post-transfusion blood chemistries
Renal and hepatic chemistries
Minimized donor exposure
Similar conclusions for AS-3 study (2000)
Jain R, Jarosz C. Safety and efficacy of AS-1 red blood cell use in neonates. Transfusion and Apheresis Science. 2001;24(2):111–115.
Compared to storage in CPDA-1, found no clinical or laboratory evidence that AS-1 RBCs had any deleterious effects
Several studies over the years that looked at the transfusion of red cells
One study—in 1996, by Strauss et al compared SMALL-VOLUME transfusion of red
cells in neonates stored in AS-1 <42 days from single donor vs SMALL VOLUME trnasufison of red cells stored in CPDA-1 stored <7 days
No significant differences in:
Pre-transfusion and post-transfusion blood chemistries
Renal and hepatic chemistries
Similar conclusions for AS-3 study done by strauss in 2000
Another study in 2001, specifically looked at the Safety and efficacy of SMALL VOLUME transfusions of AS-1 red blood cells in neonates
using rbc storage in CPDA-1 as their control, there was no clinical or laboratory evidence that AS-1 RBCs had any deleterious effects (renal/hepatic toxicity)
J Pediatr : 215–219

24. Josephson CD, Friedman D, Pizzini DS, et al.
Variability in Preparation, Storage, and Processing of Red Blood Cell Products for Extremely Low Birth Weight Infants: A Blood Bank Survey for the Transfusion of Prematures (TOP) Trial
Josephson CD, Friedman D, Pizzini DS, et al.
For small volume transfusions (< 20 ml/kg)
11/29 sites exclusively use CPD/CPDA storage solutions
6/29 sites use only additive solutions
12/29 sites use a combination of solutions.
17/22 sites, make aliquots from a RBC unit until expiration
6/29 sites do not use dedicated units until expiration; they select another donor when the unit ages 5 to 28 days old
Abstract Presented in April 2015
For small volume transfusions (< 20 ml/kg), 11/29 sites exclusively use CPD/CPDA storage solutions, whereas 6/29 sites use only additive solutions (e.g., AS-1). 12/29 sites use a combination of solutions. Dedicated donor units are supplied by 22/29 sites. Most, 17/22 sites, make aliquots from a RBC unit until expiration. Six of 29 sites do not use dedicated units until expiration; rather, they select another donor when the unit ages 5 to 28 days old; the most frequent age to switch is at 14 days.

25. RBC transfusion in neonates
For small-volume transfusions:
Aliquots of same parent bag in AS to minimize donor exposure
AS-3 over AS-1 if concern about mannitol
AS-5?
Over the years, lots of studies have shown that the additive solutions are safe and efficacious for small volume transfusions in neonates
Neonates requiring multiple small volume transfusions can have aliiquots made from the same parent bag stored in A and used up until the day of expiration to minimize donor exposure
AS-3 can be chosen over AS-1 for neonatal transfusions if there is a concern over the mannitol content which is present in AS-1 but not in AS-3—however the literature suggests that both are safe to use in small volume transfusions in neonates
No specific studies using AS-5 in small volume transfuiosn in neonates
For large volume transfusions—the use of AS is not as well established.

26. Large volume transfusions in neonates
Evidence for use of AS is not well established
No randomized controlled trials
Abstract from Eder et al. at CHOP – “Comparison of CPDA vs. AS Red Cell Transfusion to Infants on ECMO”
AS-1 & AS-3 tolerated as well as CPDA-1 units
Comparable post-transfusion lab values (Hct, Na, K, glc, Ca)
Evidence for use of AS in large volume transfusions such as ECMO, RBC exchange is not well established
- No randomized controlled trials
Abstract from Eder et al. at CHOP showed that AS-1 and AS-3 units were tolerated as well as CPDA-1 units in infants on ECMO, with comparable post-transfusion lab values—that is none were statistically significant

27. Current VUMC practice of selecting a different AS for pediatric versus adults?

28. Questions?