Deformability of transfused rbc and in vitro hemolysis.
The elongation index (EI) of rbc was evaluated as a species-independent quantitative surrogate marker of the structural changes that occur during storage. Guinea pig rbc stored from 0 to 28 days were evaluated over a range of shear stresses from 0–20 Pa by osmotic gradient ektacytometry. The EI of guinea pig rbc at maximum shear stress was decreased by 12% over 14 days of storage and by 31% over 28 days of storage. Ektacytometry of human stored rbc is shown as EI compared with shear stress on days 2, 28, and 42 (Figure A). Data indicate that under approved storage conditions for 42 days human and guinea pig rbc show similar responses to osmotic stress on day 2. While, membrane rigidity increased at differing rates, human storage at day 28 was comparable to guinea pig storage day at 14, and human storage at day 42 was comparable to guinea pig storage at day 28 (Figure A). As a result, in our model, guinea pig blood stored for 28 days was determined acceptable to approximate human blood stored for 42 days (referred to herein as old blood), and guinea pig blood stored for 21 days was evaluated for the purposes of elucidating temporal storage-dependent effects in vivo. The mean accumulation of extracellular Hb from in vitro hemolysis during storage increased from 0.41 mg/ml (0.22% of the total Hb) after leukocyte reduction to 1.50 mg/ml (~1.0% of the total Hb) at the time of transfusion (Figure B).
In vitro deformability of stored rbc.
Transfusion of longer-term storage rbc leads to sustained intravascular hemolysis.
The extent of hemolysis in the different treatment groups after 80% transfusion was followed for 24 hours to evaluate (a) hematocrit (Hct), (b) plasma Hb, and (c) size-exclusion chromatography of Hp-sequestered plasma Hb. Basal Hct in treatment groups was 35.6% ± 1.2% blood after 2 days of storage (referred to herein as new blood), 34.0% ± 0.82% old blood, and 37.3% ± 0.82% old blood plus Hp. Similar to basal levels, 24-hour Hct was 35.4% ± 1.71% after new-blood transfusion. Old blood (with or without Hp) transfusion resulted in a significant (P < 0.05, compared with new blood and baseline) Hct reduction of 8.3% (29.0% ± 2.42%) and 6% (28.0% ± 2.18%), respectively (Figure A). Pooled plasma from the new blood group showed no Hb accumulation relative to nontreated animals (NTs) (Figure B). In contrast, significant plasma Hb accumulated after old-blood transfusion with and without Hp coinfusion. Interestingly, Hb released in the old blood group was bright red (Figure B) or predominantly in the ferrous Hb (HbFe2+) form (86%), with mean area under the curve from 0 to 24 hours (AUC0–24h) values for total Hb, HbFe2+, and ferric Hb (HbFe3+) equal to 4,567 μM•h•ml–1, 3,911 μM•h•ml–1, and 656.5 μM•h•ml–1, respectively. This result suggested an ongoing level of hemolysis, coupled with rapid extravascular distribution and clearance over the 24-hour evaluation period (Figure C). Conversely, Hp coadministration led to Hb-Hp complex formation after transfusion, and, as a result of the much longer half-life of the complex compared with that of free Hb, the heme-oxidized Hb-Hp complex did accumulate over time (Figure D). The half-life of the complex is prolonged compared with the short half-life of free Hb because renal filtration of Hb (which is responsible for the rapid elimination of non-Hp-bound Hb) is blocked. Additionally, saturation of the CD163-related macrophage Hb-Hp clearance system by supraphysiological levels of Hb-Hp complex may lead to its accumulation within the circulation and delay elimination from plasma in our model. This was demonstrated by a darker red/brown color in plasma (Figure B), indicative of increased HbFe3+ (38%) as well as increased circulating total Hb-Hp (AUC0–24h = 31,464 μM•h•ml–1), HbFe2+-Hp (AUC0–24h = 19,388 μM•h•ml–1), and HbFe3+-Hp (AUC0–24h = 12,076 μM•h•ml–1). The accumulation of HbFe3+-Hp over time is shown in Figure E. Further confirmation of circulatory Hb-Hp complex formation after old blood administration (with or without Hp) is demonstrated by size-exclusion chromatography of plasma prior to and at 4, 16, and 24 hours after transfusion (Figure F). Data demonstrate that old-blood transfusion without Hp coadministration leads to cell-free Hb in plasma over a 24-hour period (chromatography peak coeluting with Hb standard at 18.5 minutes, 405 nm). Conversely, coadministration of Hp with old blood captured Hb within a Hb-Hp complex (chromatography peaks coeluting with Hb-Hp standard at 13.5 to 15 minutes, 405 nm). By 24 hours, a small free Hb peak was detectable in most plasma samples, indicating the beginning of oversaturation of coinfused Hp. Transfusion of new blood demonstrated a small total plasma Hb exposure over the 24-hour collection period (AUC0–24h = 73.4 μM•h•ml–1), which appears to be attributable to the initial period of transfusion.
In vivo Hb exposure after blood transfusion with or without Hp.
Stored rbc transfusion induces functional and structural vascular changes that are attenuated by Hp coadministration.
The vasculature is the first organ system exposed to Hb, degradation products of rbc lysis, and to Hb-Hp complexes. Exposure to free Hb is known to elicit an increase in blood pressure (20
). Hb exposures have also been associated with cardiovascular toxicity in certain species, manifested as early-onset (within 24 hours) myocardial lesions (27
). Therefore, we examined the vascular effects of new and older storage blood transfusion with or without Hp.
This study demonstrates that new-blood transfusion does not alter baseline systolic (70.1 ± 1.2 mmHg), diastolic (45.7 ± 5.1 mmHg), or mean (53.8 ± 0.54 mmHg) arterial blood pressure. Conversely, old blood transfusion significantly increased (P < 0.05) systolic (23.4% ± 10.5%), diastolic (14.7% ± 5.64%), and mean (17.9% ± 5.87%) arterial blood pressure over basal values. This effect was transient (1–2 hours); however, the coadministration of Hp with old-blood transfusion attenuated the blood pressure response to that observed at baseline and with new blood administration (Figure A).
Transfusion-related vascular changes.
Previous work has suggested a strong association between Hb and NO in blood pressure regulation during hemolytic states (16
). Our data are in general agreement with the NO depletion hypothesis; however, our comparative analysis of plasma NO consumption, NO metabolite plasma levels, blood pressure, and vascular damage over time as well as across different treatment groups (old blood with or without Hp) suggest a dissociation among these parameters (Supplemental Figures 5 and 6; supplemental material available online with this article; doi:
). Plasma from animals transfused with old blood with or without Hp demonstrated equal NO consumption at 1 hour after transfusion (Supplemental Figure 5A). At this time point, the maximal hypertensive response was observed in the animals transfused with old blood, but no blood pressure changes occurred in the Hp coinfusion group. In parallel with continuous Hb accumulation over time, NO consumption activity of plasma did increase, and significantly more NO consumption was measured with plasma sampled at 24 hours after transfusion (Supplemental Figure 5A) when blood pressure values approximated baseline. These ex vivo data are supported in vivo by the finding that NO metabolites decreased over time in all animal groups that were transfused with old blood and dose escalations of Hp (Supplemental Figure 5B), regardless of the presence or absence of a transient blood pressure response (Supplemental Figure 5C). In agreement with these findings, stopped flow kinetic data demonstrated nearly identical second-order rate constants for the dioxygenation reaction of oxy-Hb (k′ox
, NO = 18.8 μM–1
) and oxy-Hb-Hp (k′ox
, NO = 15.9 μM–1
) (Supplemental Figure 5D). The second-order rate constants for NO binding to met-Hb (k′NO(Fe3+)
= 6.9 × 103
) and met-Hb-Hp (k′NO(Fe3+)
= 6.8 × 103
) were also found to be similar (Supplemental Figure 5E). The effects of old blood plus increasing Hp doses on vascular changes are shown in Supplemental Figure 6. Taken together, the vascular protective activity of Hp treatment cannot be explained by a simple biochemical mechanism that would involve attenuated NO reactivity of the complex.
To explore other factors that may contribute to acute blood pressure attenuation after older blood administration, we analyzed Hb and Hb-Hp oxidative states over time in our model. Attenuation of Hb-induced blood pressure response has been suggested in reports that show that oxidized (Fe3+
) Hb is associated with increased viscosity (30
) and decreased reactivity with NO (31
). In Hp-treated animals, oxidized Hb (Fe3+
) in the Hb-Hp complex began to accumulate in the plasma at 8 hours, 138 ± 3.00 μM (38% of total), and reached a maximum of 239 ± 48.0 μM (45% of total) at 16 hours (Figure E). However, within the critical early time period after transfusion, when a hypertensive response can be observed in the non-Hp-treated animals, there was apparently no excess of Fe3+
Hb in the Hp-treated animals.
In this study, the root of the aortic arch was evaluated for transfusion-related injury. New-blood transfusion did not show abnormal changes in aorta (Figure , B–E). However, old-blood transfusion was associated with coagulative necrosis, in some animals extending from the luminal tunica intima to deep within the tunica media (Figure , B–E). This observation was attenuated by Hp coinfusion with old blood (Figure , B–E). Iron deposition was observed in the perivascular regions around the vasa vasorum supplying the aortic tunica adventitia and within the connective tissue after transfusion with old blood with or without Hp but not after transfusion with new blood (Supplemental Figure 4, A–C). Regions containing iron also showed colocalized immunoreactivity for HO-1 and CD163, which indicates the accumulation of peripheral blood monocytes/macrophages (Supplemental Figure 4, A–C). Taken together, these observations suggest that Hb exposure resulting from in vivo hemolysis may play a causative role in the vascular abnormalities observed after old-blood transfusion and that Hp could be effective at limiting vascular toxicity despite the increased circulation time of the Hb-Hp complex. To explore the Hp dose-dependent protective effect, we evaluated attenuation of vascular (aortic root) injury after transfusion of old blood with Hp at doses of 100 mg, 300 mg, and 900 mg (Supplemental Figure 6A). The response to increasing Hp doses was quantified by histopathological scoring of aortic root sections (Supplemental Figure 6B). Our data indicate a general improvement in old blood–induced aortic root injury with increasing Hp dose. Images in Supplemental Figure 6A show representations of normal, abnormal, and coagulative necrotic vascular tissue from representative groups. As shown in Supplemental Figure 6B, nontransfused animals and animals transfused with new blood demonstrated 90%–95% normal aortic root vasculature. Conversely, transfusion with old blood demonstrated 22% ± 4.5% (normal), 56% ± 8.2% (abnormal), and 22% ± 6.0% (necrotic) regions. This was unchanged with a 100 mg dose of Hp; however, increasing doses to 300 mg Hp prevented necrotic tissue injury, while 900 mg Hp decreased necrotic as well as abnormal tissue regions, such that 53% ± 12% were of normal appearance and only 47% ± 13% were of abnormal appearance, with no necrotic regions observed.
Timing of vascular tissue recovery.
In this nonlethal model, guinea pigs were otherwise normal and healthy absent the transfusion. After transfusion, in the vasculature, coagulative necrotic regions observed in the 28-day-old blood group developed collagen formation that accounted for 61% ± 9.8% of the aortic root by 48 hours after initial assessment. This was significantly different than collagen formation in the aortic root of animals transfused with new blood, 27% ± 5.0%, and those transfused with old blood plus Hp, 37% ± 9.0% (Figure , D and E).
Renal proteomics — protein profiling of the predominant Hb clearance organ after blood transfusion.
Representative gross morphology images of kidneys 24 hours after transfusion with new blood, old blood, and old blood plus Hp are depicted (Figure , A–C). Animals transfused with new blood demonstrated normal gross morphology; conversely, animals transfused with old blood demonstrated consistent dark red and black discoloration, particularly within cortical regions (6 out of 6 animals transfused). Coinfusion of Hp with old-blood transfusion attenuated this effect, presumably by preventing renal decompartmentalization of Hb. We screened for quantitative protein changes related to Hb exposure/metabolism, oxidative stress, and parenchymal tissue injury 24 hours after transfusion in kidneys. In 4 independent mass spectrometry experiments (with n = 4 animals per treatment group), we identified 1,554 proteins in at least 2 out of 4 experiments with a false discovery rate of less than 1% at peptide and less than 5% at the protein identification level. Quantitative estimates of protein abundances were derived from iTRAQ analysis. The graph in Figure D shows all the proteins that were identified in at least 2 out of 4 experiments. Each protein’s relative abundance across the 3 treatment groups (compared with NTs) is shown as a single line. The superimposed box plots represent the fraction of proteins that were found to be overrepresented in the new and old blood (with or without Hp) groups. These data show that a number of proteins are overrepresented after transfusion with old blood, but not new blood, and that coadministration of Hp can attenuate these changes (Figure D).
Initial tissue screening — gross pathology and proteomic profiling.
The most extensively changed proteins are summarized in Table , and a complete list of the proteomic data is provided in Supplemental Table 1. We have manually screened the literature to assign the old blood–associated proteins to functional categories (Figure ), in particular those related to Hb/heme metabolism, oxidative stress, and renal physiology/tubular reabsorption. Group differences in relative quantitation of Hb-α globin and Hb-β globin as well as Hb metabolic pathway proteins, such as heme oxygenase-1 (HO-1), biliverdin reductase B, and ferritin-heavy/light chains, were observed, indicating extensive heme exposure of the kidneys of animals transfused with old blood. Hp coinfusion with old blood attenuated accumulation of Hb-α globin and Hb-β globin and most heme-associated metabolic enzymes, while new-blood transfusion showed no changes in these protein levels. The potential impact of old-blood transfusion–associated Hb exposure on renal physiology is demonstrated by the accumulation of several plasma proteins that are typically reabsorbed and degraded by tubular epithelial cells (i.e., cystatin C). Hp coinfusion with old blood attenuated the increases in the accumulation of tubular reabsorbed proteins, while new-blood transfusion showed no changes in these protein levels (Figure ).
Quantitative analysis of the kidney proteome of animals transfused with new blood, old blood, and old blood plus Hp relative to the tissue proteome of untreated guinea pigs
Renal tissue proteomic analysis.
Hb release from stored rbc after transfusion contributes to renal pathology that is attenuated by Hp coinfusion.
Hb accumulation in the urine of animals transfused with old blood was observed after 24-hour collections; however, this was not observed in animals transfused with new blood or old blood plus Hp (Figure A). Renal exposure to Hb was further confirmed by tissue staining for iron accumulation, total renal iron, and globin chain tissue staining (Figure , B and C). Renal iron increased significantly (P < 0.05, old blood compared with new blood) at 24 hours after old-blood transfusion to 15.9 ± 2.4 μg per 100 mg tissue compared with 12.2 ± 0.92 μg per 100 mg tissue (new blood) and 13.4 ± 0.76 μg per 100 mg tissue (old blood plus Hp) (Figure D). The proteome pattern also indicated overabundance of some proteins that are controlled by the principle oxidative stress transcription factor Nrf-2, suggesting enhanced oxidative stress in the kidneys of animals transfused with old blood (Table , see noted Nrf-2–associated proteins). Immunofluorescence and Western blot analyses confirmed increased nuclear accumulation of Nrf-2 in renal tubules after old-blood transfusion and, to some extent, in animals transfused with old blood plus Hp compared with animals transfused with new blood and NTs (Figure , E and F). HO-1 expression after old-blood transfusion was 8-fold greater than that after new-blood transfusion and 2-fold greater than that after old blood plus Hp transfusion (Figure G). These data suggest that Hb/heme exposure is attenuated but not entirely blocked by Hp coinfusion. Together with the renal proteomic profiling data, these findings support the interpretation that Hb exposure and subsequent activation of metabolic, antioxidant, and circulatory protein accumulation are significantly enhanced in the old blood group compared with the old blood plus Hp and new blood group.
Renal Hb exposure, metabolic activation, and oxidative stress.
Renal histopathology of animals transfused with old blood revealed dilated proximal and distal tubules, consistent with nephrosis and tubular degeneration typically associated with tubular dysfunction (Figure A). Significant elevations in serum creatinine from NT levels (0.38 ± 0.019 mg/dl) to a 7-fold increase (3.13 ± 0.88 mg/dl) in the old blood group were observed at 24 hours. Serum creatinine in old blood plus Hp (0.45 ± 0.028 mg/dl) and new blood (0.44 ± 0.023 mg/dl) was unchanged from that in NTs (Figure C). Histopathological changes were not evident in the animals transfused with new blood or old blood plus Hp (Figure A).
Timing of renal tissue recovery.
Renal cortical injury was rapid and robust after massive transfusion of 28-day-old blood. However, these acute changes also resolved after cessation of rbc hemolysis, reflecting the strong regenerative potential of the kidney in response to acute injury. In particular, renal proximal and distal tubular dilation returned toward normal appearance by 48 hours after initial assessment (Figure B), and serum creatinine decreased accordingly from 3.1 ± 0.88 mg/dl to 0.9 ± 0.24 mg/dl (Figure C). Taken together, these data suggest that Hb release from rbc can contribute to acute renal failure caused by massive transfusion of older stored blood and that Hp can function as an effective therapeutic to attenuate toxic responses to within a physiologically manageable range.
Posttransfusion hemolysis, vascular, and renal effects of intermediate storage time rbc.
We additionally evaluated the effects of blood transfused after an intermediate storage period of 21 days (Figure ). The exposure (AUC0–24h) to extracellular Hb after 80% transfusion of 21-day-old blood was 865 ± 90 μM•h•ml–1. This was approximately 5-fold less than the 24-hour Hb exposure after 28-day-old blood transfusion but significantly greater than Hb exposure after new-blood transfusion (Figure A), and 24-hour renal excretion of Hb remained evident (Figure B). H&E staining of aortic root tissue 24 hours after transfusion (Figure C) indicated that 21-day-old blood transfusion resulted in significant damage. The areas of abnormal and necrotic appearing tissue were similar with 28-day-old blood and were also found to be associated with iron accumulation in the adventitia (Figure C). The effect of renal Hb clearance noted in 24-hour urine collections demonstrated the same extent of renal cortical iron deposition compared with that in 28-day-old blood (Figure E, bottom). Renal iron per 100 mg of tissue was significantly greater in the kidneys transfused with 21-day-old blood (15.9 ± 1.9 ng/100 mg tissue) and 28-day-old blood (17.5 ± 0.71 ng/100 mg tissue) than in those after new-blood transfusion (9.5 ± 1.2 ng/mg tissue) kidneys (Figure F). However, in contrast to transfusion of 28-day-old blood, which results in acute kidney failure, these changes appeared to represent subclinical findings, since serum creatinine was unchanged from basal levels in the group transfused with blood stored for 21 days (data not shown).
Cardiorenal response to 21-day-old blood transfusion.
In summary, the cumulative Hb exposure and tissue damage found after transfusion of blood stored for 21 days was significant compared with that after new-blood transfusion but less severe than the changes observed after old blood (28 days storage) transfusion. Therefore, a storage time–dependent component appears to contribute to the blood transfusion–associated adverse effects in our model.
The impact of in vivo (after transfusion) versus in vitro (before transfusion) hemolysis.
As shown in Figure , a small fraction of rbc do undergo hemolysis during storage, and in vitro hemolysis within the storage bag has previously been proposed as a possible factor in the pathophysiology associated with stored-blood transfusion (16
). We have therefore performed 2 sets of experiments to dissect the contribution of in vitro (before transfusion) versus in vivo (after transfusion) hemolysis in our model.
A group of animals were dosed with a bolus infusion of purified guinea pig Hb to match the peak plasma heme concentration (approximately 300 μM) achieved after old-blood transfusion. Since we found supernatant storage solution typically contained low Hb concentrations after 28 days of storage, this experiment mimics a hypothetical condition to test the effects of Hb resulting from severe in vitro hemolysis occurring in a storage bag. Results demonstrated no remarkable changes to gross vascular and renal morphology or histopathology previously shown (Figure , D and E). Additionally, no temporal changes were observed in serum creatinine levels in these animals (data not shown). These results suggest preinfusion hemolysis is not a predominant cause of the tissue damage observed in our old-blood transfusion model.
In another set of experiments, washed 28-day-old blood was transfused and resulted in a plasma Hb AUC0–24h of 2,045 ± 369 μM•h•ml–1. This value was approximately 2-fold less than the Hb exposure after nonwashed 28-day-old blood transfusion (Figure A). The pooled post-wash supernatant (6 total washes, final wash was clear) is shown as an inset in Figure B. Importantly, serial ektacytometry analysis of rbc before and after the washing procedure shows an increase in deformability. Prior to transfusion (i.e., after PBS washing) the cell population was more deformable than the original old blood, suggesting that the repeated cycles of centrifugation and resuspension removed both extracellular Hb and some Hb from nondeformable cells (Figure B). This observation is likely a result of mechanical destruction of the most fragile rbc during the wash procedure and indicates that experiments with washed rbc may in fact underestimate the true level of in vivo hemolysis after transfusion of nonwashed old blood. The 24-hour blood sample obtained from transfused guinea pigs (n = 4) demonstrated rbc with normal deformability. Aortic root changes after transfusion of 28-day-old washed blood are shown in Figure C. Tissue morphology within the aortic root demonstrated similar changes as those found after nonwashed old-blood transfusion (Figure D). The gross morphology of kidneys after transfusion with washed 28-day-old blood showed a darkened renal cortex (Figure E). Renal iron per 100 mg of tissue was significantly greater in the kidneys after transfusion with washed 28-day-old blood (19 ± 2.2 ng/100 mg tissue) and the 28-day-old nonwashed blood (17.5 ± 0.71 ng/100 mg tissue) when compared with new-blood transfusion (9.5 ± 1.2 ng/100 mg tissue) kidneys (Figure G). Histopathology of renal cortical tissue revealed abnormal morphology of proximal and distal tubules; however, tissue was absent of dilated tubules observed after nonwashed 28-day-old blood transfusion (Figure F, top). Compatible with the less severe morphologic changes after washed old-blood transfusion, we did not detect significant changes in posttransfusion creatinine levels.
Cardiorenal response to 28-day-old blood transfusion before and after washing.
In summary, these 2 sets of experiments suggest that the 2 components of in vitro and in vivo hemolysis may both contribute to the cumulative pathophysiology of stored-blood transfusion. However, bolus exposure to free Hb that could accumulate in the storage bag is apparently not sufficient to induce gross pathologic changes. In contrast, transfusion of washed rbc can mimic the general pattern of pathology observed after large-volume transfusion of old blood, even though a significant loss of fragile rbc does occur during the washing procedure and may limit correct estimations of in vivo hemolysis upon old-blood transfusion. Therefore, we assume that in vivo posttransfusion hemolysis is the main component responsible for tissue Hb exposure and pathology in our model.