Non-DEHP Container Bacterial Proliferation Examined

Purpose of the Non-DEHP Red Blood Cell Concentrates Study

A previous study in Vox Sanguinis evaluated the impact of non-di(2-ethylhexyl) phthalate (DEHP) blood bags on the in vitro quality of red blood cells and plasma. Investigators in Canada have now released the results of a new study that examined, “whether [the] proliferation of transfusion-relevant bacteria in di(2-ethylhexyl) terephthalate- (DEHT-) phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGSM) red blood cell concentrates (RBCCs) was different from bacterial growth in DEHP-saline-adenine-glucose-mannitol (SAGM) units, impacting product safety.”

The authors of the latest study appearing in Vox Sanguinis on the merits and performance of non-DEHP containers explained that, “[p]aired ABO-matched whole blood units were collected into DEHT/PAGGSM whole blood bag sets. On the same day as collection, paired whole blood units were pooled into a DEHP-free polyvinyl chloride (PVC)/citrate bag and then split evenly into one DEHT/PAGGSM bag and one DEHP/SAGM whole blood set from which the anticoagulant had been removed. Whole blood was stored overnight at room temperature, and leukoreduced RBCCs were produced on day one post collection from each of the two whole blood units using a top/bottom RBC filtration process.” For the study, [p]aired DEHT/PAGGSM and DEHP/SAGM RBCC units were inoculated with one of four transfusion-relevant bacteria: the fast-growing facultative Gram-negative Yersinia enterocolitica PEI-A-105 and Serratia liquefaciens PEI-A-184, slow growing facultative Gram-positive Listeria monocytogenes PEI-A-199, and the aerotolerant anaerobe Gram-positive Cutibacterium acnes BPN-BT-19195. Units were spiked to a final bacterial load of ~100 CFU/mL for the facultative species and to a target load of ~1000 CFU/mL of C. acnes, as this bacterium survives but does not proliferate in RBCCs. [On day two] post collection, spiked units were stored at 1–6°C for 43 days and sampled on days 0, 7, 14, 21, 28, 35 and 43 for bacterial enumeration.” The researchers “performed [the study] in triplicate]” on all the bacterial species.

Findings Regarding Non-DEHP Containers

The study found that, “in vitro quality parameters including h[e]moglobin and h[e]matocrit were not statistically significantly different between DEHP/SAGM and DEHT/PAGGSM (non-DEHP) units (p=0.889 and p=0.224, respectively. The mean cell volume was significantly higher in DEHP/SAGM RBCCs compared to the DEHT/PAGGSM (non-DEHP) units (p=0.024). H[e]molysis and residual white blood cells (rWBCs) were also significantly higher in DEHT/PAGGSM RBCCs in comparison with DEHP/SAGM units (p=0.000 and p=0.011). [A]ll DEHP/SAGM and DEHT/PAGGSM RBCCs produced for the study had acceptable in vitro quality results on day one as per Canadian Standards Association requirements, and were deemed suitable for comparing bacterial viability and growth between the two types of units.”

Additionally, the researchers noted that, “[f]or Y. enterocolitica and S. liquefaciens, there was no significant difference in the bacterial growth rate between DEHP/SAGM and DEHT/PAGGSM RBCCs (p=0.95 and p=0.87, respectively). Both species grew to a bacterial load of approximately 10⁷ CFU/mL by day seven of RBCC storage, a load that is considered clinically significant. L. monocytogenes decreased more in DEHT/PAGGSM (non-DEHP) RBCCs than in DEHP/SAGM RBCCs from day 0 to day 7 of storage, resulting in significantly different growth curve slopes (p=0.03). However, after day seven, the bacterium proliferated in both types of RBCCs, reaching similar counts (~10⁷ CFU/mL) on day 43 of storage. C. acnes counts remained at approximately 10³ CFU/mL until the end of RBCC storage, with no significant differences observed between DEHT/PAGGSM and DEHP/SAGM RBCC units (p=0.08).

Conclusion

The paper concluded that, “there is no increase in the safety risk of RBCC storage in DEHT containers with PAGGSM compared to DEHP/SAGM stored-RBCCs. [Our] results showed that the risk posed by DEHT/PAGGSM RBCC units to transfusion patients is not different from that posed by current units stored in DEHP/SAGM [for] Y. enterocolitica and S. liquefaciens, [species that] been implicated in septic transfusion reactions involving contaminated RBCCs. Interestingly, slow-growing L. monocytogenes had a sharp decrease in bacterial counts in DEHT/PAGGSM (non-DEHP) RBCC units from days 0 to 7 of storage; however, the bacterial load was comparable in both DEHT/PAGGSM and DEHP/SAGM RBCCs at the end of storage, indicating a similar safety risk for transfusion patients. The reduction in bacterial counts during early storage in DEHT/PAGGSM RBCC is intriguing and could be strain-specific or could be due to an effect of the plasticizer in combination with the change in the additive solution. [Overall,] our study, the first to provide bacterial safety data in DEHP-free blood bags, supports the substitution of DEHP bags with alternative plasticizers for RBCC storage without compromising the safety of transfusion patients in terms of bacterial contamination risk.” The authors also acknowledged that their, “[work] could be complemented by testing other bacterial species that can degrade phthalates, such as Pseudomonas fluorescens. Furthermore, it would be interesting to run a similar comparative study in RBCCs stored in containers that have other phthalate-free plasticizers such as di-isononyl-cyclohexane-1,2-dicarboxylate (DINCH) and/or n-butyryl-tri-n-hexyl citrate (BTHC), which have also been shown to be promising candidates to replace DEHP in blood collection and storage containers.”

Citation: Ramirez-Arcos, S., Kou, Y., Kumaran, D., et al. “Bacterial proliferation is comparable in red blood cell concentrates stored in DEHT/PAGGSM and DEHP/SAGM containers.” Vox Sanguinis. 2026.