Evolution of adverse changes in stored RBCs

Bennett-Guerrero et 10.1073/pnas.0708160104.XXYYYYY103.

Supporting Information

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SI Table 1
SI Figure 5
SI Methods
SI Figure 6
SI Figure 7
SI Figure 8




SI Figure 5

Fig. 5. Fractional content of metHb (A), deoxyHb (B), and oxyHb (C) in stored RBCs as a function of time. Note that the y axis maximum is 1% for metHb and 100% for deoxyHb and oxyHb. The sum of the metHb, deoxyHb, and oxyHb fractions was ³99% at each timepoint.

SI Figure 6

Fig. 6. SNO-Hb, related NO adducts, and vasoactivity of stored RBCs: individual data. (A-C and E) Total Hb-bound NO (A), Hb[Fe]NO (B), SNO-Hb (C, a calculated value equal to total Hb-NO minus Hb[Fe]NO), and RBC membrane SNO (E) were determined by the photolysis-chemiluminescence (PC assay). (D) RBC (total) SNO was determined by the CO-saturated copper cysteine (3C assay). (F) Vasoactivity represents the percentage decrease in tension induced by RBCs in the bioassay (% vasorelaxation). Because of the complexity of the membrane SNO assay, samples were assayed only at selected time points. Individual (symbols) and median (horizontal bars) data points are shown. Unprocessed samples (black) were assayed immediately (0 h) and, for some parameters, after a 3-h delay, in addition to assays at the indicated times after processing was begun (colored points, beginning at 3 h). P values represent comparison between values in RBCs assayed immediately (0 h) vs. 3 h later (in unprocessed samples for A-C and F). No significant change from 3 h to 6 weeks was observed for any of these variables in processed samples.

SI Figure 7

Fig. 7. RBC deformability (elongation index for two representative shear stress levels as a function of storage time): individual data. Individual (symbols) and median (horizontal bars) data points are shown at 3 (Left) and 30 Pa (Right). Unprocessed RBC samples (black) were assayed immediately (0 h) and processed samples at the indicated times after processing (colored points, beginning at 3 h). P values represent significance for change over time.

SI Figure 8

Fig. 8. Cytokine concentrations (plasma IL-6 and plasma IL-8) as a function of storage time. Data are shown as median with 25th and 75th percentiles. No significant increase over time was observed. All samples measured for plasma IL-1b assay and plasma TNF-a were below the level of detection (data not shown).

Table 1. Demographics

Mean ± SD (range) are shown as appropriate.

*Upper limit of normal is 6.0%.

SI Methods

Eligibility Criteria. Inclusion criteria were: male and female, healthy, Caucasian (to reduce the possibility of enrolling a sickle hemoglobin carrier) (1), and aged 18-50 years. Exclusion criteria were: any medical condition that would make blood donation unsafe; subjects who would refuse the administration of blood products for religious or other reasons; weight <110 pounds; prior history of significant adverse reaction during donation of blood or phlebotomy; known clinically significant hematological or clotting disorder; possibility of pregnancy (consistent with the AABB screening process, no pregnancy test was done); donation of blood within 8 weeks before this study; inadequate RBC mass as defined by hematocrit £38% or hemoglobin (Hb) £12.5 g/dl; temperature >99.5°F or visible signs of active infection; elevated glycohemoglobin (Hb A1C) consistent with the presence of diabetes; hypertension defined as a systolic blood pressure >180 mm Hg or diastolic blood pressure >100 mm Hg; and use of tobacco products within 1 month before blood donation.

Blood Donation and Processing. Subjects were asked to consume nothing by mouth after midnight before the donation. On arrival to the donation site (DUMC Post Anesthesia Care Unit), using aseptic technique and a blood pressure cuff, »500 ml of whole blood was collected from a peripheral vein directly into a Pall Medical Leukotrap WB System containing 70 ml of CP2D anticoagulant (containing 0.0156 M citric acid, 0.1227 M sodium citrate, 0.016 M monobasic sodium phosphate, and 0.257 M dextrose). After leukocyte depletion and centrifugation, packed RBCs were resuspended in 110 ml of Nutricel [additive solution-3; AS-3] preservative solution. Each unit of RBCs was stored in a monitored and alarmed refrigerator at 1-6°C. Aliquots of RBCs were withdrawn by using a sterile docking device (Sterile Tubing Welder TSCD model SC-201A; Terumo Medical Corp, Somerset, NJ) and transferred on ice to the appropriate laboratories for analysis at: 3, 8, 24, and 96 h, and 1, 2, 3, 4, and 6 weeks.

Assays. Overview. The following 26 variables were measured: blood culture and blood gases including pH, Hb O₂ saturation (SO₂), and partial pressures of O₂ (pO₂) and carbon dioxide (pCO₂); concentrations of the cytokines IL-1b, IL-6, IL-8, and tumor necrosis factor a (TNF-a), extraerythrocytic (cell-free) Hb, potassium, glucose, lactate, calcium, magnesium, and chloride in the storage medium; the concentration of 2,3-DPG and ATP in RBCs; NO derivatives in RBCs including SNO-Hb (assessed by using two methods; see below), total Hb-bound NO, Hb[Fe]NO and membrane SNO, and physiological and functional properties of RBCs including deformability, adhesion, vasoactivity, and surface expression of phosphatidyl serine (PS).

RBC vasoactivity assay. Briefly, the descending thoracic aortas were harvested from adult New Zealand White rabbits after killing by CO₂ inhalation. Aortic rings (3-4 mm in width) were mounted on stainless steel wires in tissue baths containing Krebs-Henseleit buffer (pH 7.4) bubbled with 21% O₂/5% CO₂/balance N₂. Isometric tension was measured and recorded continuously. Functional endothelial integrity was assessed in each ring by exposure to acetylcholine (10^-7 M) after preconstriction with phenylephrine (range, 10^-8 to 10^-5.5 M final concentration, titrated to produce ~2 gm final active tension). Hypoxia was established by switching the gas source to 5% CO₂/0% O₂/95% N_2. The pO₂ was measured by using an O₂ electrode (Mettler Toledo), and the gas bubbling rate was adjusted as necessary to achieve a pO₂ of 7 ± 2 mmHg. Vascular responses to air-exposed, washed RBCs (final hematocrit 0.4%) in hypoxia were determined.

RBC SNO-Hb, Hb[Fe]NO, and membrane SNO assay: photolysis-chemiluminescence (PC). Briefly, plasma and WBCs were removed by centrifugation, and the remaining RBCs were exposed to air and washed twice with excess PBS (PBS; containing 0.1 mM diethylenetetraaminepentaacetic acid (DTPA, as acid), final pH, 7.4). For each time point, a sample of washed RBCs was set aside, frozen, and stored at -80°C for later batch analysis to determine RBC ATP concentration and total RBC SNO concentration by the 3C method (see below). For the PC method, RBCs were lysed with 0.1 mM DTPA (in deionized H₂O, final pH, 6.92) and the Hb partially purified by passing the lysate over a G-25 Sephadex column. Paired Hb samples were treated with PBS with or without HgCl₂ (0.6 mM final) and injected into the PC device. Hb-bound NO concentrations in the absence (representing total Hb-bound NO) and presence of HgCl₂ (representing Hb[Fe]NO) were determined by extrapolation against a standard curve generated daily by using GSNO (8-250 nM; calculated SNO-Hb represents the difference between these two measured values. For membrane SNO assays, a similar procedure was applied to RBC membranes prepared as described (1, 2); this assay was applied only at a limited number of prespecified time points. Under the conditions we used, the PC assay registers little or no signal from nitrite standards, and because samples are desalted before assay, residual nitrite injected is in the low-nanomolar range and thus could not account for the NO equivalents measured (T.J.M., unpublished observation). In addition, photolytic yields of NO from nitrate under these conditions are <<1%, in either the presence or absence of added thiols (M.A.M., H.Z., and T.J.M., unpublished observations).

RBC vasoactivity and SNO-Hb levels in unprocessed blood. For analysis of fresh samples by PC, venous blood (10 ml) was obtained from eight additional healthy human volunteers meeting the eligibility criteria; demographic data for subjects in this substudy were similar (data not shown). Samples were immediately analyzed for RBC membrane SNO, SNO-Hb, Hb[Fe]NO, total Hb-bound NO levels, and RBC vasoactivity (0 h), avoiding the delays associated with large blood volume collection, distribution, and processing required to prepare additive-solution RBCs. Blood samples were collected directly into a proportionally equivalent volume of CP2D. For 0-h analysis by 3C, fresh venous blood (3 ml) was drawn from 10 additional healthy volunteers meeting the eligibility criteria. Samples were collected into heparinized tubes and assayed immediately (0 h) by 3C for total RBC SNO content.

Total RBC SNO content: Reduction in carbon monoxide-saturated copper/cysteine (3C). Frozen RBCs in PBS (see above) were thawed before batch analysis, and whole-cell SNO content was determined as described (3). Samples were frozen for <2 weeks before analysis; in pilot studies, neither freezing/thawing nor sustained frozen storage (-80°C) altered the fidelity of the 3C assay on similarly processed samples (data not shown). In this assay, SNOs are selectively converted to NO in a cuprous chloride- and carbon monoxide-saturated cysteine solution; reductively released NO is then detected via chemiluminescent reaction with ozone. Sample SNO content was indexed to total Hb concentration and expressed as a molar ratio of SNO/Hb. SNO signal measured by this technique is entirely eliminated by sample treatment with mercuric chloride. Validation experiments using the 3C method demonstrated that GSNO standard curves did not differ in the presence and absence of (lysed) RBCs added to the reaction chambers in amounts equivalent to those used in analysis of SNO content in subject RBC samples (3).

We deliberately chose not to apply an alternative SNO-Hb assay, based on the use of a complex chemical mixture including triiodide, ferricyanide, and acidified sulfanilamide, because of (i) an unacceptably high level of NO-scavenging by Hb under typical conditions, (ii) destruction by the acidified sulfanilamide of most Hb-bound SNO, and (iii) disturbance of FeNO:SNO-Hb equilibria by ferricyanide (4, 5).

RBC deformability assay (LORCA). RBC deformability was measured by using a laser-assisted optical rotational cell analyzer (LORCA; Mechatronics Instruments, Hoorn, The Netherlands) according to the manufacturer's guidelines and as described (6, 7). Briefly, an aliquot of blood was added to polyvinyl-pyrrolidone (Mechatronics). A thin layer of this RBC suspension was sheared between two concentric cylinders at nine different shear stress levels (0.3 to 30 Pa) over 4 min at 37°C. Faster rotation of the outer cylinder (cup) leads to increasing shear stress that causes RBC deformation (elongation). The laser beam diffraction pattern is detected with a video camera and analyzed by a computer. Each "measurement" represents a mean and standard deviation of 50 measurements at one of the nine shear stress levels. RBC deformability is expressed as the Elongation Index. A priori, we defined two main levels of shear stress as 3 and 30 Pa (8), with 3 Pa representing a clinically relevant level of shear stress in the microcirculation (9-11).

Cytokine assays. For cytokine analyses, sample aliquots were removed from the storage medium of the RBC units at the indicated time points. RBCs were pelleted by centrifugation, and the supernatant was removed and frozen at -80°C for later batch analysis. Cytokine concentrations were quantitated by ELISA and performed by R & D Systems (Minneapolis, MN) on thawed samples.

RBC ATP content. Batch analysis for ATP content was conducted using the luciferin technique (ATP Bioluminescent Assay kit; Sigma, St. Louis, MO) after thawing and deproteinizing frozen RBC samples that had been washed and stored in PBS (see above) (12, 13).

RBC 2,3-DPG concentration. 2,3-DPG concentration in stored RBCs was determined by using the detection kit from Roche Diagnostics (Mannheim, Germany) and with monitoring at 340 nm on a Shimadzu UV160U spectrophotometer. Briefly, sample aliquots were extracted at the indicated time points, the RBC samples were deproteinized according to the manufacturer's instructions, and the samples frozen and stored at -80°C for later batch analysis.

Extraerythrocytic(free) Hb assay. As described (13), an aliquot of frozen RBC storage medium was thawed, diluted in PBS, and treated with excess dithionite and nitrite (converting all Hb species present to Hb[Fe]NO), and absorbance was determined at 417.5 nm (the Soret peak for Hb[Fe]NO) by using UV-visible spectrophotometry.

PS exposure. Exposed PS on the RBC surface membrane was measured by treating RBCs with commercially available phycoerythrin-labeled annexin V (BD Biosciences) in the presence of calcium and measuring cell-associated fluorescence in a Becton-Dickinson FACScan. Controls run with each experiment included unstained cells, cells stained with annexin V only, and RBCs treated with calcium ionophore.

Statistical Methods and Data Integrity. No formal sample size calculations were performed given the descriptive nature of this study. Our objective was to collect data from 15 volunteers. Electronic data were entered into a computer database by the Cato Research (CATO) Data Management group. For each parameter, the individual subject-by-time profiles (by using values after processing) were created and compared by using a split-plot formulation of a repeated-measures ANOVA. Comparisons for changes over time were determined from the ANOVA by using successive time contrasts and linear contrasts. These models were fit for observed values. Differences between the fresh (time point 0) and stored blood values were determined by using the exact Wilcoxon two-sample test. A two-sided P value of <0.05 was considered statistically significant.

The authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the manuscript as written.

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