P-CB-2 - Reticulocyte-rich RBC Units Exhibit Distinct Morphological and Biochemical Changes During Cold Storage

Sunday, October 26, 2025

12:15 PM PT

Presenting Author(s)

Krystalyn E. Hudson, PhD (she/her/hers)

Associate Professor
Columbia University Irving Medical Center
New York, New York

Disclosure information not submitted.

Background/Case Studies: Donor blood units contain variable levels of reticulocytes, which we have previously shown to enhance red blood cell (RBC) alloimmunization. Given that reticulocytes retain active mitochondria, we hypothesized that reticulocyte-rich (RR) units may exacerbate oxidative stress during cold storage, leading to increased hemolysis and altered RBC morphology.

Study

Design/Methods: Reticulocytosis was induced in GFP+ mice via alternate-day phlebotomy (100 µL/day) for two days. On day 4, blood was collected by cardiac puncture. Pooled GFP+ blood was mixed 1:1 with GFP− (naïve) blood to create RR units (~10% reticulocytes). Control units were composed solely of GFP− blood (~2% reticulocytes). Blood units were stored at 4°C and sampled at baseline, day 3, 7, and11 for morphological analysis (Amnis ImageStream®) and metabolomic profiling (LC-MS/MS).

Results/Findings: Reticulocyte frequencies increased over 11 days of storage in both control and RR units, rising from 1.8% to 2.5% in controls and from 9.8% to 18.1% in RR units. Reticulocytes in both groups retained mitochondria (78% in control vs. 89% in RR), and RR units exhibited elevated levels of tricarboxylic acid (TCA) and urea cycle intermediates, consistent with ongoing metabolic activity. At baseline, the projected surface area (PSA) was greater in RR units (46.1 µm² vs. 44.9 µm²), and these cells also showed increased phospholipid content. After 11 days of storage, PSA declined in RR units (45.9 µm² vs. 47.2 µm² in control). Within RR units, GFP+ cells demonstrated a higher proportion of large cells ( >50 µm²) and a progressive shift from intermediate-sized (35–50 µm²) to small-sized ( < 35 µm²) cells after cold storage. In contrast, GFP− cells exhibited a shift from intermediate to large cell populations, with no increase in small cells, suggesting selective hemolysis of intermediate-sized GFP− RBCs during storage. Hemolysis was higher in RR units (19.8% vs. 12% in control) stored for 11 days.

Conclusions: These findings show that reticulocyte-rich (RR) units undergo distinct biochemical and morphological changes during storage. Metabolically active reticulocytes, retaining mitochondria, may accelerate oxidative damage and hemolysis in co-stored mature RBCs—supporting a "bad apple" hypothesis. Shifts in GFP+ and GFP− RBC subpopulations suggest that intermediate-sized cells are particularly vulnerable to damage or loss. Increasing reticulocyte frequency over time may reflect selective depletion of older, fragile RBCs. Donor reticulocyte content thus influences storage lesion development and may impact RBC storage quality, transfusion efficacy, and alloimmunization.