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Blood, Vol. 113, Issue 5, 1158-1166, January 29, 2009
Loss of red cell chemokine scavenging promotes transfusion-related lung inflammation
Blood Mangalmurti et al.
113: 1158
Supplemental materials for: Mangalmurti et al
Flow cytometric Analysis of purified murine erythrocytes
1 × 105 purified murine erythrocytes were suspended in FACS buffer and labeled with PE conjugated TER-119 (BD Biosciences-Pharmingen, San Jose, CA, USA) or FITC conjugated CD45 (BD Biosciences-Pharmingen, San Jose, CA, USA). Isotype control antibodies were PE conjugated rat IgG2b,κ, and mouse FITC conjugated IgG2a,κ (BD Biosciences-Pharmingen, San Jose, CA, USA). FACS analysis was performed as described above. Limulus Amebocyte Lysate Assay
PBS, colloid solution-Hextend® (Hospira, Inc.), and erythrocytes were tested for the presence of lipopolysaccharide (LPS) by Limulus Amebocyte Lysate (LAL) assay (Cambrex Corp., East Rutherford, NJ, USA). Measurement of Temperatures
A standard infant thermometer was used to obtain auricular temperatures. The highest temperature of 3 separate readings was recorded for each mouse at each time point. Measurement of pH, K+, 2,3 DPG, Lactate, and free Hemoglobin in fresh whole blood or erythrocyte concentrates
Whole blood was obtained from C57/BL6J WT mice as previously described. Purified erythrocytes were washed 3× with cool sterile PBS and stored in 50 ml polypropylene Falcon tubes. Fresh human blood was obtained from healthy volunteers via venipuncture and collected into heparinized glass phlebotomy tubes. Human RBCs were isolated as previously described and stored in AS-5 (1:3 parts; Adsol, erythrocytes). Banked human blood was obtained from the Institute for Transfusion Medicine (Pittsburgh, PA, USA). Lactate, pH, and potassium levels in both murine and human blood samples were determined by i-STAT clinical analysis (Heska, Loveland, CO). Free Hgb levels were determined by colorimetric assay via Quantichrom detection kit from BioAssay (BioAssay, Hayward, CA, USA). 2,3-DPG levels were determined by using the detection kit from Roche diagnostics (Roche, Manheim, Germany).
Files in this Data Supplement:
- Table S1. Effect of storage on RBC concentrates from C57/BL6 WT mice and human banked blood (PDF, 17.4 KB)
- Figure S1. Isolation and processing of erythrocytes (JPG, 34.5 KB)
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(A) Erythrocyte concentrates were purified from murine whole blood and labeled with PE conjugated TER-119 or FITC conjugated CD45. FACS analysis revealed Ter-119+/CD45− cells; demonstrating a > 99% pure erythrocyte population. (B) Endotoxin units/ml were measured in PBS, Hextend® (HEX) and red blood cell (RBC) transfusates. Reference standard (LPS) is shown for comparison.
- Figure S2. Temperature and plasma cytokines following tail vein LPS injection (JPG, 43.8 KB)
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(A) Auricular temperatures were decreased from baseline at both 2 and 10 hours after tail vein LPS injection (p<0.00001). (B) Plasma chemokines and cytokines were elevated at 2 and 4 hours after LPS injection when compared with controls (P=0.001) for MIP-2, KC, IL-6, and TNFα at both timepoints. Data represent 2 independent experiments at each timepoint, N=6-12 animals in each group.
- Figure S3. Plasma chemokine concentrations 2, 4, and 16 hours following transfusion of 1–2 d RBC in endotoxemic mice (JPG, 33.9 KB)
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(A) MIP-2 is significantly elevated at 2 hours in the 1–2 d RBC transfused group, but not at 4 or 16 hours after transfusion when compared with hextend (P = 0.044, P = 0.878, and P = 0.101 at 2, 4, and 16 hours). (B) Plasma KC was not elevated at 2 or 4 hours but was elevated at 16 hours after erythrocyte transfusion when compared with hextend transfusion; P= 0.095, P = 0.158, and P = 0.035 at 2, 4, and 16 hours. Data represent 2 independent experiments at each timepoint, N=6–12 animals in each group.
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