Research Review: Mapping the Kinetics of Damage During Long-Term RBC Storage

The Problem

Storing cells for long periods of time is often a tricky situation. Exactly how long can you keep them in storage before they’re no good?

Prolonged red blood cell (RBC) storage can cause severe damage to them. The cells can undergo significant structural and biochemical changes that compromise their morphology, function, and DNA integrity. Collectively, these alterations are referred to as “storage lesion.” ¹

For applications from DNA extraction for PCR to life-saving blood transfusions, using healthy, functional red blood cells is critical. While red blood cells can be stored long term for up to 42 days, “younger” cells around 18 days old are typically preferred in transfusions involving packed red blood cells (PRBC) to avoid complications such as acute lung injury and high mortality.^2-5 When it comes to DNA extraction, even younger (ideally fresh, unfrozen) cells are preferred to isolate as much high-quality DNA as possible to bolster efficiency and accuracy of downstream assays, such as PCR and next-generation sequencing.

Understanding the processes involved in storage-induced damage, and the precise circumstances they occur under, is key to optimizing conditions for long-term red blood cell storage for both the research lab and the clinical lab.

To find out how RBCs incur damage during extended periods of containment, Kozlova et al. set out to track structural changes in the cells during storage, specifically the kinetics of cytoskeletal damage, in an attempt to unravel the precise steps involved in the process.

Study Design

To investigate cytoskeletal changes during red blood cell storage, the researchers used atomic force microscopy (AFM) to evaluate the cytoskeleton mesh in PRBC samples from several different time points during storage.

They used the AFM images to calculate parameters of cytoskeletal configuration, such as pore number and pore size. From this information, the investigators developed a kinetic model that allowed for the time-dependent determination of damage.

Findings and Implications

The researchers found that long-term PRBC storage leads to

• Rupture of cytoskeletal filaments
• Merging of small pores into larger ones
• Filament thickening (cluster formation or fusion) and membrane stiffness

There are two key stages in the cytoskeletal damage process: filament rupture and cluster formation. Filament rupture was the first stage of damage, which began at days 9-10; the rate of the process reached a maximum by the end of week 2. Cluster formation began on days 18-21, when proteins began to cluster and filaments thickened; the maximum rate was reached around day 30.

While the investigators didn’t look into the exact mechanisms driving these changes, it is well understood that oxidative stress is involved in cytoskeleton damage during PRBCs storage.

Nevertheless, through this work, the researchers provided important insight into the steps involved in the deterioration of cytoskeletal integrity in stored PRBCs. This is important information for optimizing storage conditions to help preserve the form and function of PRBCs.

Technical Highlight

During the course of their studies, the researchers relied on dependable reagents from MP Bio. They prepared PRBC ghosts to visualize the cytoskeletal network by using phosphate buffered saline (PBS) from MP Bio to first do a slow wash out of the red blood cells from the storage solution (citrate-phosphate-dextrose (CPD)-saline, adenine, glucose and mannitol (SAGM)).

After performing several more washes (at 1,500 rpm) followed by hemolysis with a solution of 0.09% NaCl in distilled water, they mounted the ghosts onto glass slides coated with MP Bio’s poly-L-lysine and evaluated them in an AFM open liquid cell.

References

1. Lelubre, C. et al. Relationship between red cell storage duration and outcomes in adults receiving red cell transfusions: a systematic review. Critical Care 17: R66 (2013).
2. García-Roa M, Del Carmen Vicente-Ayuso M, Bobes AM, et al. Red blood cell storage time and transfusion: current practice, concerns and future perspectives. Blood Transfus 15: 222-31 (2017).
3. Xu Z, Zheng Y, Wang X, et al. Stiffness increase of red blood cells during storage. Microsyst Nanoeng 4: 17103 (2018).
4. Weinberg JA, McGwin G Jr, Vandromme MJ, et al. Duration of red cell storage influences mortality after trauma. J Trauma 69: 1427-32 (2010).
5. Clinical Practice Guidelines From the AABB: Red Blood Cell Transfusion Thresholds and Storage. JAMA 316(19): 2025-2035 (2016).