Photo by Elise Amendola
Certain methods of manufacturing red cell concentrates (RCCs) may be less damaging than others, according to research published in Vox Sanguinis.
The study showed that damage-associated molecular patterns (DAMPs) in stored blood differ according to the process and materials used to collect or prepare RCCs for transfusion.
Researchers believe this discovery could help reduce adverse reactions in transfusion recipients and potentially impact how blood is collected around the world.
To conduct this study, the researchers compared RCCs collected at blood donation centers in the US and Canada. The team examined the influence of manufacturing methods on the levels of mitochondrial (mt) DNA and extracellular vesicle (EV) DAMPs in RCCs.
“Working with the American team at Blood Systems Research Institute was key to this research because of the wide variations in blood manufacturing processes present in the US,” said Jason Acker, PhD, of Canadian Blood Services’ Centre for Innovation in Edmonton, Alberta.
“In countries like Canada, where there is a national blood service, manufacturing methods are largely standardized, so it is difficult to compare various methods. But blood collection in the US is characterized by dozens of independent blood centers that use a variety of available manufacturing processes. The Americans provided the variations we needed to measure red cell damage and to ascertain whether it can be attributed to different manufacturing methods.”
Manufacturing methods
The researchers evaluated 87 RCCs prepared using 9 different methods, outlined in the following table.
| Name | Processing method | Collection/
manufacturing method |
Anticoagulant/
additive solution (AS) |
Leuko-reduction (LR) method | LR temperature and timing |
| FenBC (n=6) | Semi-automated whole blood (WB) processing
following overnight (O/N) hold at 18–24 °C: Buffy coat (BC) method |
Collection set: Fenwal CGR6494B, Quad OptiPure RC 9SBT WB 500 ml Component processing: Compomat G4 | Citrate phosphate dextrose (CPD)/saline adenine glucose mannitol (SAGM) | Filtration of RCC | 20–24°C within 24 h of the stop bleed time |
| MacoBC
(n=6) |
Semi-automated WB processing
following O/N hold at 18–24°C: BC method |
Collection set: Macopharma LQT 7291
LX Leucoflex LCR-Diamond Quadruple Bottom and Top System, WB 500 ml Component processing: Compomat G4 |
CPD/SAGM | Filtration of RCC | 20–24°C within 24 h of the stop bleed time |
| FenWBF
(n=6) |
Semi-automated WB processing:
WB filtration method |
Collection set: Fenwal CGR8441B, Quad
PackPure WB 500 ml Component processing: Compomat G4 |
CPD/SAGM | Filtration of WB | 1–6°C within 72 h of the stop bleed time |
| MacoWBF
(n=6) |
Semi-automated WB processing:
WB filtration method |
Collection set: Macopharma Leucoflex MTL1 Quadruple Top and Top System, WB 500 ml
Component processing: Compomat G4 |
CPD/SAGM | Filtration of WB | 1–6°C within 72 h of the stop bleed time |
| FenMAN
(n=12) |
Manual WB processing | Fenwal 4R1582 Double Blood-Pack Unit
500 ml, with Flex-Excel Red Cell Filter |
CPD/AS-1 | Filtration of RCC | Room temperature (RT) within 8 h of stop bleed time |
| FenMAN-non-LR
(n=12) |
Manual WB processing | Fenwal 4R1587P Triple Blood-Pack Unit 500 ml | CPD/AS-1 | Not applicable | Not applicable |
| Alyx (n=15) | Apheresis | Fenwal Software: 3.0; 4R5720 Alyx 2RBC-LR Kit | Acid citrate dextrose A (ACD-A)/AS-1 | Filtration of RCC | RT post-
collection |
| MCS+ (n=12) | Apheresis | Haemonetics; Software Rev H or L; 0832F-00, 2RBC filtered | CP2D/AS-3 | Filtration of RCC | RT if <8 h/
cold if >8 h post- collection |
| Trima (n=12) | Apheresis | Terumo BCT; Software 6.0.6; Trima Accel 80500 kit | ACD-A/AS-3 | Filtration of RCC | RT if <8 h/ cold if >8 h post-
collection |
Results
For all RCCs, the researchers assessed the levels of mtDNA and the number and cell of origin of EVs on storage days 5 and 42.
They observed a 100-fold difference in mtDNA levels between the different methods.
The highest mtDNA levels were in the non-leukoreduced RCCs, followed by the MCS+ and Trima apheresis RCCs. The mean levels were 5.3 x 105 copies/µL, 1.3 x 105 copies/µL, and 1.2 x 105 copies/µL, respectively.
The lowest mtDNA levels were seen with the semi-automated methods. The mean levels ranged from 3.8 x 103 copies/µL to 5.9 x 103 copies/µL.
The researchers also saw a 10-fold difference in EV levels between the different methods.
The team detected red blood cell-derived CD235a+ EVs in fresh RCCs, which increased in most RCCs over the storage period (but not for FenBC and MCS+).
Platelet-derived CD41a+ EVs were highest in non-leukoreduced and Trima RCCs and did not change significantly during storage.
White blood cell-derived CD11b+ and CD66b+ EVs were low in most RCCs (though not in Trima and FenMAN-non-LR RCCs), and their levels did not significantly change during storage.
White blood cell-derived CD14+ EVs were negligible in fresh RCCs but increased in several RCCs during storage (FenMAN, Alyx, MCS+, and Trima).
Next steps
“There must be more testing of the apheresis collections equipment, blood bags, leukoreduction filters, and other variations in manufacturing methods to determine what single element or combination of elements in the various red blood cell manufacturing processes result in high levels of DAMPs and why,” said Michael Busch, MD, PhD, of Blood Systems Research Institute in San Francisco, California.
“We also need to understand how mitochondrial DAMPs are involved in adverse reactions to red blood cell transfusions,” added Sonia Bakkour, PhD, also from Blood Systems Research Institute.
“Some recently published studies on platelet components link high levels of mitochondrial DAMPs to adverse transfusion reactions. We need to see if DAMPs have similar adverse effects on recipients of red blood cell transfusions.”
“We think that our research could lead to finding the best way to manufacture red blood cells,” Dr Acker noted.
“It’s clear now that manufacturing methods matter. We . . . are keen to explore what’s in the blood bag or in the filters or in the tubing, for example, that can be minimized or eliminated, improving the outcome in patients who receive blood transfusions.”
