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Study authors (from left)
Brant M. Wagener,
Jean-Francois Pittet,
and Rakesh Patel.
Photo from University
of Alabama at Birmingham

Free heme plays a key role in the adverse effects associated with massive transfusion of stored red blood cells (RBCs), according to researchers.

Experiments in a mouse model of trauma hemorrhage revealed a greater risk of mortality from bacterial pneumonia in mice that received transfusions of blood stored for 14 days, rather than fresh blood.

This greater risk was dependent upon free heme, which is released from RBCs during storage and upon transfusion.

In a study of human trauma patients, researchers found the amount of heme was proportional to the amount of blood transfused.

Brant M. Wagener, MD, PhD, of University of Alabama at Birmingham, and his colleagues detailed these findings in PLOS Medicine.

In the mouse model of trauma hemorrhage, the researchers resuscitated mice using either fresh blood (stored for 0 days) or blood stored for 2 weeks. (A 2-week storage of mouse blood approximates storage of human RBCs for 42 days.)

Two days after transfusion, the mice were challenged by instilling the lungs with the bacteria Pseudomonas aeruginosa.

Mice that received the stored blood had a significant increase in bacterial lung injury, as shown by higher mortality, and increased fluid accumulation and bacterial numbers in the lungs.

The researchers identified the connection between free heme and infection susceptibility/severity in 2 ways.

First, Pseudomonas aeruginosa-induced mortality was completely prevented by the addition of hemopexin, a scavenging protein that removes free heme from the blood.

Second, adding an inhibitor of toll-like receptor 4 (TLR4), or genetically removing TLR4 from mice, also prevented bacteria-induced mortality. Free heme—which is known to induce inflammatory injury to major organs in diseases like sickle cell or sepsis—acts, in part, by activating TLR4.

The researchers also found that transfusion with stored blood induced release of the inflammation mediator high mobility group box 1 (HMGB1). But an anti-HMGB1 antibody protected mice from bacteria-induced mortality.

The anti-HMGB1 antibody also restored macrophage-dependent phagocytosis of Pseudomonas aeruginosa in vitro.

Tissue culture experiments had revealed that free heme inhibits macrophages from ingesting Pseudomonas aeruginosa, and the addition of free heme increases permeability in endothelial cells.

Finally, in a 16-month study, the researchers found that human trauma-hemorrhage patients who received large amounts of transfused blood were also receiving amounts of free heme sufficient to overwhelm the normal amounts of hemopexin found in a person’s blood.

The researchers said this work underscores the need to confirm whether the storage age of transfused RBCs correlates with increasing levels of free heme after transfusion. The team would also like to establish whether patients with low ratios of hemopexin to free heme have a greater risk for adverse outcomes after massive transfusions.

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