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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Anesth Analg. Author manuscript; available in PMC 2014 February 18.
Published in final edited form as:
PMCID: PMC3927839
NIHMSID: NIHMS344836

Fresh and Stored Red Blood Cell Transfusion Equivalently Induce Subclinical Pulmonary Gas Exchange Deficit in Normal Humans

Abstract

Background

Transfusion can cause severe acute lung injury, although most transfusions do not appear to induce complications. We tested the hypothesis that transfusion can cause mild pulmonary dysfunction that has not been noticed clinically and is not sufficiently severe to fit the definition of transfusion-related acute lung injury.

Methods

We studied 35 healthy normal volunteers who donated one unit of blood 4 weeks and another 3 weeks before two study days separated by one week. On study days two units of blood were withdrawn while maintaining isovolemia, followed by transfusion with either the volunteer’s autologous fresh red cells (RBCs) removed 2 hours earlier or their autologous stored RBCs (random order). The following week each volunteer was studied again, transfused with the RBCs of the other storage duration. The primary outcome variable was the change in alveolar to arterial difference in oxygen partial pressure (AaDO2) from before to 60 min after transfusion with fresh or older RBCs.

Results

Fresh RBCs and RBCs stored for 24.5 days equally (P=0.85) caused an increase of AaDO2 (fresh: 2.8 mmHg [95% CI: 0.8 - 4.8; (P=0.007)]; stored 3.0 mmHg [1.4 - 4.7; (P=0.0006)]). Concentrations of all measured cytokines, except for interleukin-10 (P=0.15), were less in stored leukoreduced (LR) than stored non-LR packed RBCs; however, vascular endothelial growth factor was the only measured in vivo cytokine that increased more after transfusion with LR than non-LR stored packed RBCs. Vascular endothelial growth factor was the only cytokine tested with in vivo concentrations that correlated with AaDO2.

Conclusion

RBC transfusion causes subtle pulmonary dysfunction, as evidenced by impaired gas exchange for oxygen, supporting our hypothesis that lung impairment after transfusion includes a wide spectrum of physiologic derangements and may not require an existing state of altered physiology. These data do not support the hypothesis that transfusion of RBCs stored for >21 days is more injurious than that of fresh RBCs.

Transfusion can cause lung injury. Severe injury, transfusion-related acute lung injury (TRALI), first described in 19831 with the acronym developed in 1985,2 remains a diagnosis of exclusion in patients experiencing new acute lung injury within 6 hours after having been transfused.3 The mortality of TRALI is thought to be approximately 5% to 20%,2,4 and the Food and Drug Administration has reported that TRALI was the leading cause of transfusion-related death each year from 2005 to 2009.5 Clinical experience and the NHLBI Working Group6 definitions for this severe form of pulmonary injury, TRALI, require radiologic evidence of bilateral pulmonary edema and hypoxemia for a positive diagnosis. We conjectured that it is possible that there might be lesser pulmonary dysfunction after transfusion that had not been noticed clinically and is not sufficiently severe to fit the definition of TRALI.

In a retrospective chart review of critically ill patients, without case cohort controls, Cornet at bal. noted a decreased PaO2/FIO2 24 hrs after leukoreduced (LR) red blood cell (RBC) transfusion.7 Covin et al. reported a single case of acute transient pulmonary insufficiency after transfusion during surgery of an autologous unit of RBCs that when tested had significant lipid-priming activity.8 While antibody mediation is an accepted etiology of TRALI, the latter case report and several laboratory experiments9-12 support the additional thesis that pulmonary injury after transfusion may be caused also by infusion of active biologic substances (cytokines, phospholipids) that activate neutrophils that have been primed by an existing abnormal physiologic state. However, there are no data from prospective randomized studies in humans that sought subtle changes in pulmonary function after transfusion, while at the same time comparing any such effects after transfusion of “fresh” RBCs with those that have been stored for substantial periods of time sufficient to accumulate the implicated biologically active compounds. Some retrospective analyses of databases have noted an association of transfusion of RBCs stored for a longer time with an increased incidence of adverse clinical outcomes than with transfusion of RBCs stored for lesser periods of time.13 However, other similar analyses,14-16 including a recent larger, more carefully analyzed database of all transfused people in Denmark and Sweden, did not confirm those findings.17

Accordingly, we tested the following hypotheses: (1) that transfusion results in subclinical alterations of pulmonary function in normal humans who do not have an existing condition that causes neutrophil priming; (2) that transfused cytokines in blood components are associated with altered pulmonary function in normal humans; and (3) that transfused RBCs stored for a relatively long time compared to those stored for a lesser period produce subtle changes in pulmonary function.

Methods

To test the hypothesis that transfusion can produce relatively small, detectable changes of pulmonary function, and not only severe damage (with a low incidence), we assessed oxygenation and pulmonary dead space fraction. We assessed the latter because increased dead space fraction is an early feature of acute lung injury.18 These variables were assessed in healthy volunteers transfused with autologous stored (LR or not LR) or autologous fresh RBCs, so that results would not be affected by the presence of disease or concurrent medications.

Thirty-five healthy volunteers were studied with their signed written informed consent and with the approval of the IRB of The University of California, San Francisco (UCSF). Criteria for enrollment were that volunteers were required to be healthy as determined by history and physical examination, be nonsmokers, of age 18-35 years, have a body weight ≥ 110 lbs., an hematocrit ≥ 0.34, and be taking no medications other than for birth control.

After each volunteer gave their informed consent they were randomly allocated to one of four groups based on whether or not their donated blood was to be LR, and whether s/he would have the first study day as return of fresh or stored blood (see below). Randomization was accomplished using computerized code written for SAS version 9.1 (SAS Institute Inc., Cary, NC) using blocks of 4 volunteers. The study coordinator accessed the randomization code for one volunteer at a time. The volunteers and the investigators involved in data collection and analysis of study end-points were blinded to group allocation. The study coordinator and one physician not involved in data collection that day were not blinded for that day so that they could perform the required checks of the recipient and the units of RBCs to be transfused.

One unit of blood (450 mL) was withdrawn from each volunteer approximately four weeks before the study days, and a second unit approximately three weeks before. Blood was collected into Fenwal / Baxter bags with CPDA-1 (Fenwal Division, Baxter Healthcare Corp, Deerfield, IL) with or without (randomly allocated) an inline whole blood leukoreduction filter (Sepacell RS-2000; Asahi Kasei Kuraray Medical Co., Ltd.; Asahi Kasei Corp, Tokyo, Japan). The whole blood was separated by standard procedures in the UCSF blood bank and stored as packed RBCs (PRBCs) in the UCSF blood bank under standard conditions.

Each volunteer was studied twice, on two different days, separated by one week (Fig. 1). On each of the two study days, the volunteer rested in a semirecumbent position, with his / her head elevated at approximately 30° to 45°. The same elevation was maintained, as the angle of the lungs influences respiratory quotient (R) and dead space to tidal volume ration (VD/Vt).19 An IV cannula (16g or 18g) was placed in each arm, and a 22g cannula inserted in a radial artery after infiltration with a local anesthetic. After 10 to 15 minutes of quiet rest, the volunteer was fitted with a nose clip so that all breathing was through the mouth. On the first study day the subject had a practice session for familiarization with the equipment and procedures. During test periods (15 min [95% CI: 14-16 min] after hemodilution - blood withdrawal with maintenance of isovolemia - see below; and 66 min [95% CI: 65-67 min] after transfusion) subjects breathed room air through a rubber mouthpiece that was connected to a low-resistance valve (model 2630; Hans Rudolph, Inc., Kansas City, MO), into a Douglas bag. A low-resistance three-way valve was interposed between the large bore (43mm diameter) tubing connecting the Rudolph valve and the Douglas bag, allowing the subject to exhale through the system without collection of expired gas until stability had been reached for at least several minutes. Stability was determined by constant monitoring of mixed expired partial pressure of CO2 (CO2 sensor P-61B and CO2 analyzer CD-3A, AEI Technologies, Pittsburgh, PA). These instruments were calibrated at the start of each study day, and several times during each study day. Electrical signals were recorded using a Macintosh computer with software written by one of us (JF) for LabVIEW (version 8.5 or 8.6, National Instruments Corporation, Austin, TX). After mixed expired CO2 stabilized, expired gas was collected for 3 min in a Douglas bag that had been previously completely evacuated by use of suction.

Fig 1
Study procedures and mean times. Ninety-five percent confidence intervals of the times appear in the text.

During each test period, in addition to determination of mixed expired partial pressures of O2 and CO2, blood was sampled from the arterial cannula for measurement of blood gases (ABL 500 or ABL 800, Radiometer America, Cleveland, OH), complete blood count, cytokine concentrations (see below), hemoglobin concentration, and oxyhemoglobin saturation (OSM 3 Hemoximeter, Radiometer America, Cleveland, OH).

After initial measurements, two units (450 mL each) of blood were withdrawn through an IV cannula into a standard blood collection bag containing CPDA-1 (Fenwal Baxter). These units were not LR on either study day and were processed into PRBCs by the UCSF blood bank in standard fashion. Albumin, human 5% (Grifols, Los Angeles, CA) was simultaneously infused through the IV cannula in the other arm, to maintain isovolemia (1.1 times the volume of blood removed20). A second set of measurements was made (see above; Fig. 1) and a sample of blood was taken 15 minutes [95% CI: 14-16 min] after completion of this procedure.

After a rest period of 60 min [95% CI: 57-63 min], 2 units of autologous RBCs [approximate volume 193 mL per unit] were infused for 28 min [95% CI: 26-30 min]. On one study day the infused erythrocytes were those stored for 3 to 4 weeks, and on the other study day they were the erythrocytes withdrawn the day of the study (order of study days randomly allocated). Each volunteer was studied with both fresh and stored RBCs, on separate days. Stored erythrocytes were randomly allocated to be LR or not. On both study days, another set of measurements and collection of blood samples was performed 66 min [95% CI: 65-67 min] later.

Cytokines

We measured the following cytokine concentrations (minimal detectable concentration in pg/mL) in volunteer blood samples each time pulmonary function was assessed: interleukin (IL)-1ra (0.49), IL-1beta (0.23), IL-2 (0.13), IL-4 (0.03), IL-6 (0.2), IL-8 (0.16), IL-10 (0.17), granulocyte macrophage-colony stimulating factor (GM-CSF) (0.07), interferon-gamma (0.16), tumor necrosis factor-alpha (0.55), and vascular endothelial growth factor (VEGF) (0.23) using a Bio Rad cytokine kit (Bio Rad Laboratories, Hercules, CA) and an elisa-based assay (Luminex’s xMAP® technology; Invitrogen Corp., Life Technologies, Carlsbad, CA). These cytokines were also measured in the RBCs at time of infusion.

Primary end-point

The pulmonary gradient for oxygen - the difference between partial pressures of oxygen in the alveoli (PAO2) and arterial blood (PaO2) - (AaDO2) was assessed on each study day after isovolemic removal of two units of blood (before transfusion of PRBCs), and again after transfusion of the autologous PRBCs. The primary end-point of the study was the difference of the effect of stored autologous RBCs with that of fresh autologous RBCs on the change of AaDO2 between these two times.

The alveolar partial pressure of oxygen was calculated from the alveolar gas equation21:

equation M1

R was assumed to be 0.86.22,23

Secondary end-point: the secondary end-point was the difference in change of VD/Vt from after withdrawal of two units of blood but before transfusion to after transfusion, between stored autologous and fresh autologous RBCs.

equation M2

where PECO2 is the partial pressure of CO2 in mixed expired gas.

Statistics

Power analysis

We determined the number of volunteers to be studied by an a priori two-tailed power analysis using preliminary data from healthy patients transfused during posterior spinal surgery. The analysis, assuming a correlation of 0.5 between changes in one condition compared to changes in the second condition, determined that studying 35 volunteers would give the study a power of 0.8 with an alpha of 0.05. We had planned to study 38 volunteers to allow for 3 volunteers to withdraw before completing the full study; however, that was not necessary because all completed the study.

Data were tested for normal distribution by the Shapiro-Wilk test. Comparisons between groups and within groups (measurements made after removal of blood but before transfusion were compared with those made 66 min after transfusion) using Student’s paired-t test. For data that were not normally distributed (cytokines), comparisons were made using the Mann-Whitney U and the Wilcoxon sign-rank tests. A nonparametric test, Spearman’s rho, was used to test for correlation among cytokine concentrations. Examination of possible effects of order of experimental day (fresh or stored), AaDO2, PaO2, and VD/Vt were compared for all volunteers by Student’s paired t-test. All tests were two-sided. Statistical tests were performed using JMP (version 7.0; SAS Institute, Cary, NC) or InStat (version 3.0b for Macintosh; GraphPad Software, Inc, La Jolla, CA). Statistical significance was accepted at P≤0.05. Presented P values were not modified for analyses of multiple testing of cytokine concentrations and associations.

Data are presented as mean [95% confidence interval, CI] or median (interquartile range, IQR) unless otherwise indicated.

Results

Thirty-five volunteers completed the study; 20 transfused with LR RBCs, and 15 transfused with non-LR RBCs. There were no statistical differences for gender, age, ethnicity, height, weight, or body mass index between those transfused with LR or non-LR RBCs (Table 1).

Table 1
Demographics.

“Fresh” RBCs were transfused 1.67 [95% CI: 1.53-1.80] hours, and “stored” RBCs were transfused 24.5 [24.0-24.9] days after collection.

Pulmonary

Data from three volunteers were excluded from analysis (1 LR, 2 not LR) for AaDO2, PaO2, and VD/Vt because of calibration errors of the blood gas instrument.

  1. Primary outcome measure: Alveolar-arterial difference of partial pressure of oxygen (AaDO2): There were no differences for absolute values or change of AaDO2 between LR and non-LR RBCs at any time point on either day (transfusion with fresh or 24.5 day stored RBCs; range of P values: 0.65 to 0.99). Therefore the data from the two groups were pooled for comparison of transfusion with fresh or 24.5-day stored RBCs. Transfusion of either fresh or stored RBCs increased AaDO2. The change in AaDO2 did not differ (P=0.85) when volunteers were transfused with fresh (change: 2.8 mmHg [0.8 to 4.8 mmHg]; P=0.007) or stored (change: 3.0 mmHg [1.4 - 4.7 mmHg]; P=0.0006) RBCs (Fig. 2). The difference in AaDO2 from before to after RBC transfusion between fresh and older RBCs was 0.24 mm Hg [95% CI: -2.8 to 3.2 mm Hg]. This value is 8% of the effect size, a value substantially less than that frequently required to establish “equivalence.”
    Fig 2
    Alveolar to arterial difference for partial pressure of oxygen (AaDO2) from before (white bars) to after (horizontally striped bars) transfusion increased significantly after transfusion with red blood cells (RBCs) stored for 1.7 hrs (*P=0.007) and RBCs ...
    AaDO2 was greater (P=0.021) after transfusion of stored (7.0 mmHg [5.1-9.0 mmHg]) than after fresh (4.8 mmHg [2.4 - 7.1 mmHg] RBCs; however, this difference appears to have been influenced by a numerical, but not statistically significant, difference before transfusion (P=0.12).
  2. Arterial blood partial pressure of oxygen (PaO2): There were no differences for PaO2 between LR and non-LR RBCs at any time point on either day (transfusion with fresh or 24.5 day stored RBCs; range of P values: 0.36-0.95). Therefore the data from the two groups were pooled for comparison of transfusion with fresh or 21-day stored RBCs.
    PaO2 did not change with transfusion of fresh RBCs (change: -1.4 mmHg [-3.8 to 1.0 mmHg]; P=0.25) but decreased by a small but statistically significant amount with transfusion of stored RBCs (2.0 mmHg [-4.0 to -0.1 mmHg]; P=0.038; Fig. 3). However, the change of PaO2 did not differ between transfusion of fresh or stored RBCs (P=0.62). Sixty-six minutes after transfusion, PaO2 did not quite reach a statistically significant difference (P=0.063) between fresh (102.5 mmHg [98.9 - 106.1 mmHg]) and stored (100.3 mmHg [97.9-102.9 mmHg]) RBC groups.
    Fig 3
    Change (gray bars) of arterial partial pressure of oxygen (PaO2) from before (white bars) to after (horizontally striped bars) transfusion did not differ (P= 0.62) between the red blood cells (RBCs) of different storage duration. Data are mean, 95% confidence ...
  3. Ratio of respiratory dead space volume to tidal volume (VD/Vt): There were no differences for VD/Vt between LR and non-LR RBCs at any time point on either day (transfusion with fresh or older stored RBCs; range of P values: 0.47 to 0.88).
    VD/Vt did not change with transfusion of either fresh (change: 0.00 [-0.02 to 0.02]; P=0.84) or stored (0.01 [-0.01 to 0.03]; P=0.47) RBCs (Fig. 4). There was no statistical difference between these tiny changes comparing transfusion of fresh RBCs with that of stored RBCs (P=0.76).
    Fig 4
    Change (gray bars) of ratio of respiratory dead space to tidal volume (VD/Vt) from before (white bars) to after (horizontally striped bars) transfusion did not differ (P= 0.76) between the red blood cells (RBCs) of different storage duration. VD/Vt did ...

The order of experimental day (fresh or stored) did not affect changes caused by transfusion for AaDO2 (P>0.99), PaO2 (P=0.67), or VD/Vt (P=0.77).

Cytokines

All cytokine concentrations were analyzed statistically by the Wilcoxon sign-rank test, because nearly all were not distributed normally.

Transfused RBCs (in vitro): Concentrations of all measured cytokines were less in LR RBCs than fresh RBCs (all P<0.0001). All cytokines in stored LR RBCs were less than in stored non-LR RBCs (all P<0.0001 except GM CSF P=0.008), except for IL-10 (P=0.15).

in vivo: nearly all in vivo cytokine concentrations after blood withdrawal (“dilution”) did not differ between fresh and stored days or between those who were to receive LR or non-LR RBCs. Transfusion of LR RBCs on the fresh day did not change in vivo concentration of any cytokine in the LR group. In the non-LR group on the fresh day transfusion of non-LR RBCs did produce a few statistically significant, but relatively modest changes in some cytokine concentrations. Transfusion of stored LR RBCs did not result in in vivo changes of most cytokines except for small statistically significant increases of IL-6 (median [IQR]: 0.3 pg/mL [0.1-1.2] to 1.5 pg/mL [0.4-3.5]; P=0.018), IL-10 (3.4 pg/mL [0.6-5.3] to 5.3 pg/mL [3.7-8.8]; P=0.016), and VEGF (8 pg/mL [5-11] to 12 pg/mL [8-15]; P=0.014). Transfusion of stored non-LR RBCs resulted in significant, modest increases of IL-6 (0.2 pg/mL [0.1-0.9] to 2.8 pg/mL [1.2-3.2]; P=0.040), IL-8 (1.7 pg/mL [1.0-2.4] to 3.1 pg/mL [1.8-4.1]; P=0.020), and VEGF (11 pg/mL [5-16] to 18 pg/mL [13-31]; P=0.0034) concentrations. In comparing LR with non-LR RBC transfusion, both on fresh and stored days, the only significant difference was a larger VEGF in vivo concentration after non-LR RBCs than after LR RBCs (P=0.018; Fig. 5). Similarly, the only significant increase in cytokine concentration comparing the change from before to after transfusion between LR and non-LR, was for VEGF for transfusion of stored RBCs (P=0.0046).

Fig 5
Transfusion of fresh (*P=0.0046) or stored (LR: # P=0.014; non-LR: † P=0.0034) red blood cells (RBCs) increased in vivo VEGF concentrations in recipients. Vascular endothelial growth factor (VEGF) increased (“change”) more after ...

With the exception of VEGF, in vivo concentrations of each cytokine correlated with in vivo concentrations of every other cytokine (all P<0.05, most <0.0001; range of Spearman’s rho: 0.18-0.80). The only cytokines with which VEGF correlated were IL-6 (P=0.029; Spearman’s rho =0.18) and IL-8 (P<0.0001; Spearman’s rho = 0.52). VEGF was the only cytokine with in vivo concentrations that correlated with AaDO2 (P=0.018; Spearman’s rho = 0.21). The correlation was stronger when transfusion of only stored non-LR RBCs was considered (P=0.0010; Spearman’s rho = 0.38). Changes of in vivo concentration of all cytokines correlated with changes of all other cytokines (nearly all P<0.0001; range of Spearman’s rho: 0.33-0.87). No change in cytokine concentration correlated with change of AaDO2 (range of Spearman’s rho: 0.004-0.17); however, correlation of change in VEGF with change in AaDO2 nearly reached significance (Spearman’s rho = 0.21; P=0.099).

Discussion

The main findings of this study are: (1) autologous RBC transfusion produces a mild decrement in gas exchange in normal healthy adults; (2) the pulmonary effects do not differ between RBCs that are fresh or have been stored for more than 21 days; (3) the pulmonary impairment is not affected by leukoreduction of the transfused RBCs despite a larger concentration of cytokines in the non-LR stored PRBCs than in the LR stored PRBCs; (4) transfusion of autologous RBCs increased the in vivo concentrations of several cytokines; and (5) VEGF was the only cytokine tested with in vivo concentrations that correlated with AaDO2.

The data reported here demonstrate that RBC transfusion results in a subtle decrement of pulmonary gas exchange for oxygen, supporting our hypothesis that lung injury related to RBC transfusion may produce a wider spectrum in humans than has been hitherto recognized, and not only overt TRALI. RBC transfusion may cause lung injury in animal models, but the limited data in humans supporting this concept emanate primarily from severe injury (TRALI) and retrospective analyses of databases. There have been two mechanisms postulated for such injury: (1) damage mediated by antibodies in donor plasma; and (2) damage induced by biologically active compounds in stored blood or blood components. Our experimental design allowed us to test the second possibility by transfusing autologous RBCs, thus eliminating the possibility of an antibody-mediated process. We demonstrated that transfusion of autologous RBCs causes a change in pulmonary gas exchange of oxygen, albeit small and in healthy volunteers, as well as an increase of concentrations of several in vivo cytokines after transfusion, thus implicating biologically active compounds as a likely etiology. Additional evidence points to the potential of neutrophil-platelet aggregates in producing organ damage.24,25 It is possible that the effects we observed might be accentuated by a patient’s clinical condition before transfusion. Such patient risk factors for the development of TRALI have been identified recently in a large prospective observational study.26

Stored RBCs have a relatively small amount of plasma stored and administered with them. Accordingly, the number of cases and incidence of severe lung injury and death attributed to RBC transfusion has been relatively low compared to those attributed to transfusion of plasma or platelets.5 Thus, the second etiology would seem more likely for many cases where RBC transfusion may be implicated as causing lung injury. Data from laboratory experiments are not unanimous as to whether RBCs alone without a “first hit” and primed neutrophils can cause lung injury.10,27-30 Our data demonstrate that RBC transfusion in healthy humans, without a first hit, and without a possible antibody-antigen mechanism can alter pulmonary function. Thus, the etiology of the changes observed in our volunteers must have been mediated by a mechanism not involving antibodies.

A potential mechanism for RBC-induced pulmonary injury is that of alteration of the pulmonary microvascular endothelium. Supernatant fluid from human RBCs stored for 3 days to 6 wks increases the permeability of human lung microvascular endothelial cell monolayers.31 VEGF has a similar action,32 and increases pulmonary vascular permeability33 increasing pulmonary edema in mice.34,35 In our experiments VEGF was the only cytokine in vivo concentrations that correlated with AaDO2. VEGF-induced increased permeability of pulmonary endothelial cells could explain our finding of increased AaDO2 with transfusion and may be a contributing factor to TRALI and transfusion-associated circulatory overload, as well. However, our finding of an association of in vivo concentrations of VEGF with AaDO2, is not a determination of a cause-and-effect relationship.

It has been more difficult to implicate RBC transfusion as a cause of human clinical pulmonary dysfunction. Retrospective database analyses have been inconsistent in attempts to find associations between RBC transfusion and morbidities, including pulmonary dysfunction or infection,7,13,14,36-41 prompting the initiation of several prospective randomized trials, including a large multicenter National Institutes of Health-sponsored effort (“RECESS”).42

Our results show that fresh RBCs and those stored for ≥21 days produced statistically equivalent subtle deficits in pulmonary gas exchange for oxygen. While proving our hypothesis that acute lung dysfunction after transfusion can have a subtle as well as severe component, we also simultaneously demonstrated that transfusion of stored RBCs did not produce more impairment than did transfusion of fresh RBCs in the volunteers that we studied. If cytokines are involved, this finding may suggest that the in vivo cytokine response could be of greater importance than the cytokine concentrations in the supernatant fluid of stored RBCs.

The lack of differences of arterial PO2 after transfusion would make clinical detection of such a subtle dysfunction difficult, because AaDO2 is not calculated frequently. The changes in gas exchange of oxygen that we observed after transfusion of two units of RBCs are sufficiently small that they would be difficult to observe in a clinical setting. This may help explain why a minor form of the disorder may not have been clinically recognized previously. Additionally, it is possible that transfusion of a larger volume of RBCs might produce a greater impairment of gas exchange that would be sufficient for clinical detection. Even if AaDO2 were to be calculated in patients, the absolute values of AaDO2 that we observed after transfusion, although representing statistically significant increases, were small and were within the normal range.43 Unless others examine changes in AaDO2 (as did we), rather than absolute values, the absolute values of AaDO2 would not be regarded as abnormal. Furthermore, AaDO2 is least in the supine position because of the lesser distance from the bottom to the top of the lung, and thus there is lesser variation of V/Q.19,23 Because most patients would likely be supine if AaDO2 were to be assessed, detection of an AaDO2 abnormality would be even less likely. Additionally, patients breathing supplemental oxygen, but without their tracheas intubated, would have an inconstant FIO2, making accurate estimation of AaDO2 impossible. Lastly, our study design maximized the ability to detect changes by using repeated measures analysis; this design and robust analysis would not be possible in a clinical setting. However, it is possible that these changes after transfusion might be more substantial after transfusions of larger volume.

The modest increases seen in IL-6, IL-8 and VEGF in the recipients of stored LR and non-LR RBC units may reflect a mild proinflammatory effect of these units, because these cytokines are mainly associated with acute inflammatory reactions.44 VEGF has a direct effect on vascular permeability through its ability to induce changes in endothelial cell adhesion and tight junction formation.32,45 VEGF increases pulmonary vascular permeability33 and overexpression of VEGF in mice produces pulmonary edema.34,35 Mild changes in vascular permeability would likely underlie the differences seen in AaDO2 in some of the transfusion recipients.

We found that VD/Vt did not change with transfusion of either fresh or stored RBCs. Although increased VD/Vt is a feature of early acute respiratory distress syndrome18 and is associated with increased mortality in those with acute respiratory distress syndrome, VD/Vt is more sensitive to changes in pulmonary perfusion than ventilation.46 VD/Vt is less in the supine than in the standing position19 owing to nonperfused, ventilated alveoli at upper lung regions when the lungs are longitudinal. Thus, it is not surprising that the changes in AaDO2 were not accompanied by changes in VD/Vt, and implies that the changes we observed were not related to pulmonary perfusion.46

Critique

Several issues of our study deserve discussion. We did not find a difference between fresh and stored RBCs for the small, but statistically significant decrements in the primary outcome variable, pulmonary gas exchange for oxygen as assessed by AaDO2 or for PaO2. AaDO2 is a more reliable measure, because it is true gas exchange and accounts for other variables (R and PaCO2) that influence PaO2. Our study was powered to detect changes in AaDO2, but not PaO2. Similarly, it should be noted that our study design did not allow for repeated measures analysis for comparison of LR and non-LR RBC transfusion and was not powered for this variable, and thus that comparison was less robust to permit detection of any difference(s) should they exist.

There is another possible interpretation of the etiology of our finding for changes in AaDO2. In as much as the changes produced by fresh or stored RBCs did not differ, it is possible that they resulted from the augmentation of blood volume per se. The infusion of less than 300 mL of RBCs (representing approximately <7% of the volunteers’ blood volume) would have initially augmented blood volume by not more than this amount, but with time fluid shifts would have partially ameliorated towards normal both that increase and extracellular volume changes. Furthermore, all volunteers were healthy and relatively young, with compliant vessels and myocardium; the small augmentation would not likely have altered cardiac pre-load or after-load. We could have controlled partially for this variable by studying each volunteer a third time without withdrawing two units of blood, but with administration of an equivalent volume of colloid. However, pharmacokinetic differences between these colloids and RBCs would not provide an exactly equivalent control. Our assertion that the relatively small volume per se of RBC transfused did not cause the changes we noted in gas exchange is supported by the stability of the volunteers’ arterial blood pressure (4 mmHg nonstatistically significant difference) and was in keeping with blood pressure changes in similar volunteers undergoing isovolemic hemodilution, owing to changes in hemoglobin concentration and systemic vascular resistance20 (viscosity and total vessel cross-sectional area47).

Our calculations for AaDO2 included an assumption for the value of R, because we did not measure oxygen consumption. Regional lung R varies with body position and within the lung based on regional differences in VA/Q.19,23 Use of 0.86 is a reasonable estimate based on the body position of our volunteers, because in the supine position any regional V/Q variations are small and the semirecumbent position serves to lessen the differences of the erect position, and published values.22,23 Any difference between the value used and the true overall average value would have been small and have had a minor effect on calculated PAO2 and thus AaDO2. Furthermore, any difference would have been included in all calculations for AaDO2 and thus would not likely have affected the value of the primary outcome measure, the change in AaDO2.

The purpose of our study was to test whether stored RBCs could alter pulmonary function. We did not focus on mechanisms of a potential effect, because we did not know a priori whether there would be any changes with RBC transfusion. Assessments of cytokine concentrations and neutrophil priming could be a logical next step to further identify the mechanisms responsible for the changes we observed. Thus, our exploratory evaluations of potential mechanisms were limited, and further constrained by the co-variation of most cytokines. The observed correlation of in vivo VEGF concentrations and AaDO2, and the knowledge of the action of VEGF on endothelial layers and pulmonary vascular permeability are suggestive that VEGF may play a role in the small oxygenation changes we detected, but do not provide proof.

Although the magnitude of the observed changes was small, it is possible that these would be amplified with higher volume transfusions or in patients with underlying disease or with an inflammatory state with either primed neutrophils or increased pulmonary vascular permeability. This study is the first demonstration of decrement in gas exchange of oxygen after autologous RBC transfusion, and points to factors other than antibody-antigen reactions as an etiology.

We conclude that RBC transfusion causes subtle pulmonary dysfunction, as evidenced by impaired gas exchange for oxygen, supporting our hypothesis that lung impairment after transfusion includes a wide spectrum of physiologic derangements and may not require an existing state of altered physiology. These data do not support the hypothesis that transfusion of RBCs stored for >21 days is more injurious than that of fresh RBCs in healthy people. These findings may not apply to those at higher risk for pulmonary injury owing to an underlying condition or disease. Similarly, we did not concomitantly transfuse plasma or platelets, and it is possible that these might accentuate the observed RBC-induced impairment. The data suggest that VEGF may play a role in the small oxygenation changes that we detected.

Acknowledgments

Funding Supported by a Public Health Service Award from the National Heart, Lung and Blood Institute, National Institutes of Health, Grant # 1 P50 HL54476

Footnotes

DISCLOSURES:

Name: Richard B. Weiskopf, MD

Contribution: This author helped designed the study, analyze the data, write the manuscript, and review the manuscript.

Name: John Feiner, MD

Contribution: This author helped design the study, acquire the data, analyze the data, write the manuscript, and review the manuscript.

Name: Pearl Toy, MD

Contribution: This author helped design the study, write the manuscript, and review the manuscript.

Name: Jenifer Twiford, BSN

Contribution: This author helped acquire the data, write the manuscript, and review the manuscript.

Name: David Shimabukuro, MD

Contribution: This author helped acquire the data and review the manuscript.

Name: Jeremy Lieberman, MD

Contribution: This author helped acquire the data and review the manuscript.

Name: Mark R. Looney, MD

Contribution: This author helped acquire the data, write the manuscript, and review the manuscript.

Name: Clifford A. Lowell, MD, PhD

Contribution: This author helped write the manuscript and review the manuscript.

Name: Michael A. Gropper, MD, PhD

Contribution: This author helped design the study, analyze the data, write the manuscript, and review the manuscript.

The authors declare no conflicts of interest.

Contributor Information

Richard B. Weiskopf, Department of Anesthesia & Perioperative Care, University of California, San Francisco, San Francisco, California.

John Feiner, Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, California.

Pearl Toy, Department of Anesthesia & Perioperative Care, University of California, San Francisco, San Francisco, California.

Jenifer Twiford, Department of Anesthesia & Perioperative Care, University of California, San Francisco, San Francisco, California.

David Shimabukuro, Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, California.

Jeremy Lieberman, Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, California.

Mark R. Looney, Department of Medicine, University of California, San Francisco, San Francisco, California.

Clifford A. Lowell, Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California.

Michael A. Gropper, Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, California.

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