David J. Ciesla

Age of transfused blood is an independent risk factor for postinjury multiple organ failure

BACKGROUND Blood transfusion has repeatedly been demonstrated to be an independent risk factor for postinjury multiple organ failure (MOF). Previously believed to represent a surrogate for shock, packed red blood cell (PRBC) transfusion has recently been shown to result in neutrophil priming and pulmonary endothelial cell activation. We have previously observed that the generation of inflammatory mediators is related to the length of PRBC unit storage. The purpose of this study was to determine if age of transfused PRBC is a risk factor for the development of postinjury MOF. METHODS Using our prospective database of trauma patients at risk for developing MOF, we identified patients who developed MOF (MOF+) and received 6 to 20 units of PRBCs in the first 12 hours following injury. A similar cohort of patients, matched for ISS and transfusion requirement, who did not develop MOF (MOF-) were also identified. The age of each unit of PRBC transfused in the first 6 hours was determined. Multiple logistic regression was performed to determine if age of transfused blood is an independent risk factor. RESULTS Sixty-three patients were identified, 23 of whom were MOF+. There was no difference in ISS and transfusion requirement between MOF+ and MOF- groups. MOF+ patients, however, were significantly older (46+/-4.7 years versus 33+/-2.3 years). Moreover, mean age of transfused blood was greater in the MOF+ patients (30.5+/-1.6 days versus 24+/-0.5 days). Similarly, the mean number of units older than 14 and 21 days old were greater in the MOF+ patients. Multivariate analysis identified mean age of blood, number of units older than 14 days, and number of units older than 21 days as independent risk factors for MOF. CONCLUSION The age of transfused PRBCs transfused in the first 6 hours is an independent risk factor for postinjury MOF. This suggests that current blood bank processing and storage technique should be reexamined. Moreover, fresh blood may be more appropriate for the initial resuscitation of trauma patients requiring transfusion.

Cervical spine fracture patterns predictive of blunt vertebral artery injury.

BACKGROUND Aggressive screening for blunt cerebrovascular injury (BCVI) has uncovered an astonishing incidence of vertebral artery injuries (VAIs) and associated stroke rate. Stroke incidence is reduced with early recognition and prompt anticoagulation. Because of the proximity of the cervical spine and vertebral arteries, we queried whether all patients with cervical spine fractures required arteriography to rule out VAI. METHODS Four-vessel cerebrovascular angiography remains the standard screening test for patients at risk for BCVI. Patients undergoing angiographic screening for blunt cerebrovascular injuries have been prospectively followed at our regional trauma center since January 1990; however, in January 1996, we began aggressive screening based on injury patterns. RESULTS Ninety-two patients with vertebral artery injuries were identified during the study period from January 1996 to June 2002. Two patients with vertebral injuries had minor cervical fractures, a C6 body fracture and a C7 spinous process/laminar fracture; both underwent diagnostic angiography for injury mechanism. Of the 21 patients without cervical spine fracture, angiographic screening for BCVI was performed for neurologic symptoms (11 patients), basilar skull fracture (6 patients), or severe facial fractures (4 patients). Cervical spine fracture was the sole indication for VAI in 69 patients. The fracture patterns were subluxations in 38 patients (55%) or extension of the fracture through the foramen transversarium in 18 patients (26%). The remaining injuries (18%) were located in the upper cervical spine: isolated C1 arch in eight patients and C2/3 body fractures in five patients. CONCLUSION Blunt vertebral artery injury is associated with complex cervical spine fractures involving subluxation, extension into the foramen transversarium, or upper C1 to C3 fractures. Routine screening should incorporate these findings to maximize yield while limiting the use of invasive procedures.

Plasma from aged stored red blood cells delays neutrophil apoptosis and primes for cytotoxicity: abrogation by poststorage washing but not prestorage leukoreduction.

BACKGROUND Blood transfusion-particularly that of older stored red blood cells (RBCs)--is an independent risk factor for postinjury multiple organ failure. Immunomodulatory effects of RBC transfusion include neutrophil (PMN) priming for cytotoxicity, an effect exacerbated by longer RBC storage times. We have found that delayed PMN apoptosis in trauma patients is provoked by transfusion, independent of injury severity. We hypothesized that aged stored RBCs delay PMN apoptosis, but that prestorage leukodepletion or poststorage washing could abrogate the effect. METHODS Healthy volunteers each donated 1 unit of blood. One half was leukodepleted, and RBC units were processed in the usual fashion and stored at 4 degrees C. Aliquots were removed on days 1, 14, 21, and 42 and the plasma fraction isolated. Selected aliquots were washed with normal saline before plasma isolation. PMNs harvested from healthy controls were incubated (5% CO2, 37 degrees C) with unmodified, leukoreduced, or washed RBC plasma (20% plasma/80% RPMI 1640), and apoptosis assessed by morphology after 24 hours. Apoptotic index (apoptotic PMNs/total PMNs) was compared. PMN priming for superoxide release was also assessed after plasma exposure. RESULTS PMN apoptosis was delayed by RBCs stored for 21 or 42 days. Prestorage leukodepletion did not alter the effect. However, washing 42-day-old RBCs abrogated the effect. PMN priming for superoxide was provoked by stored packed RBCs in an identical pattern to delayed apoptosis. CONCLUSION Plasma from stored RBCs-even if leukoreduced-delays apoptosis and primes PMNs. The effect becomes evident at 21 days and worsens through product outdate (42 days), but may be prevented by poststorage washing. Inflammatory agents contaminating stored blood likely mediate the effect. Modification of transfusion practices (e.g., giving fresher or washed RBCs or blood substitutes) may attenuate adverse immunomodulatory effects of transfusion in trauma patients.

Stored red blood cells selectively activate human neutrophils to release IL-8 and secretory PLA2.

Packed red blood cell (PRBC) transfusion has been invoked previously with immunosuppression and increased infections, but it has now been demonstrated that stored PRBCs (>14 days) can prime PMNs and provoke multiple organ failure. Recently, the role of PMNs in the genesis of MOF has been extended to their release of inflammatory cytokines, notably IL-1, IL-8, TNFalpha, and secretory phospholipase A2 (sPLA2). We hypothesize that stored PRBCs can act as a second event via stimulating the release of inflammatory cytokines from PMNs. Isolated human PMNs were incubated for 24 h in RPMI with either 20% fresh plasma or plasma from 42 day old PRBC (day of outdate) and release of IL-8, IL-1beta, TNFalpha, and sPLA2 were measured. Plasma from stored PRBCs contained small amounts of IL-8, sPLA2, and TNFalpha (102.1 +/-5.6 pg/ml, 87.6+/-6.0 pg/ml and 9.7+/-.7 pg/ml). Levels of IL-1beta were below detection (<1 pg/ml). Day 42 PRBC plasma stimulated significant PMN release of both IL-8 and sPLA2 as compared to both control and day 0 plasma (*P < .05), but PRBC plasma did not stimulate PMN release of either IL-1beta or TNFalpha. Transfused blood is emerging as an inflammatory agent that is capable of producing PMN priming. In this study we have demonstrated that PRBC plasma selectively activates PMNs to release both IL-8 and sPLA2. Thus, transfusion of PRBCs may represent a preventable inflammatory insult via modification of both blood banking and transfusion practices.

Hypertonic saline attenuation of polymorphonuclear neutrophil cytotoxicity : Timing is everything

BACKGROUND The potential to modulate the inflammatory response has renewed interest in hypertonic saline (HTS) resuscitation of injured patients. However, the effect of the timing of HTS treatment with respect to polymorphonuclear neutrophil (PMN) priming and activation remains unexplored. We hypothesized that HTS attenuation of PMN functions requires HTS exposure before priming and activation. METHODS Isolated PMN were incubated in HTS (180 mM Na+) before L-alpha-phosphatidylcholine, beta-acetyl-gamma-O-alkyl (PAF)/N-formylmethionyl-leucyl-phenylalanine (fMLP) priming/activation, after priming, or after priming/activation. Superoxide production was measured by the reduction cytochrome c, elastase release by cleavage of AAPV-pNA, and beta2-integrin expression by flow cytometry. RESULTS HTS before priming or activation decreased beta2-integrin expression, superoxide production, and elastase release. In contrast, HTS after priming/activation augmented superoxide production and elastase release. CONCLUSION The timing of HTS is a key variable in the attenuation of PMN cytotoxic functions. Maximal attenuation of cytotoxicity is achieved before priming, whereas HTS exposure after activation augments cytotoxicity.

Blood transfusion and the two-insult model of post-injury multiple organ failure.

Neutrophils (PMNs) have been implicated in the pathogenesis of multiple organ failure (MOF). The two-insult model of MOF is based on the fundamental concept that two sequential and independent insults that are individually innocuous against the host can cause overwhelming inflammation. The in vitro PMN priming/activation sequence simulates the two-insult model. Our work has demonstrated that transfusion is an early consistent risk factor for post-injury MOF and lysophosphatidylcholines (lyso-PCs) are generated in stored blood components. Additionally, platelet-activating factor (PAF) is a key inflammatory agent produced in severely injured patients. We therefore hypothesize that two events, trauma and transfusion, enhance PMN cytotoxicity irrespective of the sequence. Superoxide (O2-) production was measured by reduction of cytochrome c, adherence to fibrinogen was assessed by the radioactivity of adherent Na2(51)CrO4 (51Cr)-labeled PMNs, and endothelial cell (EC) damage by measuring the radioactivity released from 51Cr-labeled human umbilical vein endothelial cells monolayers. Isolated PMNs were primed with buffer, PAF (2 microM), or lyso-PCs (4.5, 15, and 30 microM) followed by activation with buffer, N-formyl-methionyl-leucyl-phenylalanine (fMLP) (1 microM), PAF (2 microM), or lyso-PCs (4.5, 15, and 30 microM). Neither PAF nor lyso-PCs alone stimulated O2- production. While PAF alone caused PMN adherence, lyso-PCs alone did not allowed PMNs to adhere to fibrinogen. However, both combinations of PAF/lyso-PCs and lyso-PCs/PAF significantly augmented O2- production and PMN adherence. Furthermore, these enhanced PMN cytotoxic responses significantly caused EC damage. These findings suggest that in the scenario of the two-insult model, early or late transfusion administered following trauma can provoke PMN cytotoxicity via priming or activation, thereby increasing the risk of post-injury MOF.

Validation of Postinjury Multiple Organ Failure scores

Most multiple organ failure (MOF) scores were developed over a decade ago, but little has been done in terms of validation and to understand the differences between populations identified by each of them. Given the lack of a gold standard, validation must rely on association with objective adverse outcomes. Thus, we propose to (a) validate two widely accepted MOF scores (Denver and Marshall), examining their association with adverse outcomes in a postinjury population; and (b) compare risk factors, characteristics, and outcomes of patients identified by each score. The Denver MOF score grades (from 0-3) four organ dysfunctions (lung, kidney, liver, and heart) and defines MOF as a total score more than 3. The Marshall score grades, in addition, central nervous system and hematologic dysfunction (total of six organs on a 0- to 4-point scale). Using a prospectively collected data set, MOF was scored daily by both scores for 1,389 consecutive trauma patients with Injury Severity Score of more than15 admitted from 1992 to 2004. Risk factors, clinical outcomes (death, ventilator-free days), and resource utilization outcomes (mechanical ventilation time, length of stay in the intensive care unit) were evaluated. Both scores were associated with areas under the receiver operating characteristic curves of 80 or greater (ideal value = 100), with values for the Denver score being slightly greater (albeit not significantly) regarding prediction of most outcomes. Values of sensitivity and specificity were more than 70% for death and ventilator-free days (with the Denver score showing a consistent trend toward greater specificity), but either sensitivity or specificity was less than 70% for mechanical ventilation time and length of stay in the intensive care unit, suggesting that these scores are appropriately biased toward clinical outcomes as opposed to resource utilization. Both scores performed well, with the Denver MOF score showing greater specificity, which, coupled with its simplicity, makes it an attractive tool for both the research and clinical environments. Basic concepts of each score can probably be combined to produce an improved MOF score.

The two-event construct of postinjury multiple organ failure.

The two-event construct of postinjury multiple organ failure (MOF) is based on the fundamental concept that injury primes the innate immune system such that a second insult, during this vulnerable window, provokes unbridled systemic inflammation, resulting in organ dysfunction (Fig. 1) (1, 2). Because the neutrophil (PMN) is a pivotal early effector cell in the pathogenesis of postinjury MOF (3–5), PMN priming/activation is typically studied as the in vitro surrogate of the two-event phenomenon (6). Priming is defined as an enhanced response to a stimulus that is from previous exposure of the cell to a different agonist (7). PMN priming for cytotoxicity encompasses a wide range of physiologic responses, including superoxide anion generation, degranulation of enzymes, cytokine (IL8) and lipid mediator (LTB4) production, enhanced integrin expression (CD11b/CD18), decreased selectin expression (L selectin), reduced deformability, cellular elongation, and delayed apoptosis (8). Physiologically relevant priming agents are diverse and include platelet-activating factor (PAF), LTB4, C5a, substance P, TNF, IL8, granulocyte-macrophage-colonystimulating factor, interferon, lipopolysaccharide (LPS), influenza virus, and L selectin cross-linking and CD18 crosslinking. The objectives of this brief overview are to provide some historic perspective on the two-event hypothesis and to present supporting evidence from the clinical arena as well as the bench.

Post-hemorrhagic shock mesenteric lymph activates human pulmonary microvascular endothelium for in vitro neutrophil-mediated injury: the role of intercellular adhesion molecule-1.

BACKGROUND Splanchnic hypoperfusion is believed to be central in the pathogenesis of hemorrhagic shock-induced acute respiratory distress syndrome and multiple organ failure. Our previous work focused on the portal circulation as the conduit for gut-derived mediators of acute respiratory distress syndrome. Our current focus is the proinflammatory effects of postshock mesenteric lymph. We hypothesize that postshock lymph induces neutrophil (PMN)-mediated endothelial cell damage in an intercellular adhesion molecule-1 (ICAM-1)-dependent fashion, and devised a two-insult model to test this hypothesis. METHODS Rats (n > or = 5) underwent hemorrhagic shock (mean arterial pressure, 40 mm Hg for 30 minutes) and resuscitation (shed blood plus two times crystalloid) with lymph collection. Human pulmonary microvascular endothelial cells (HMVECs) were divided into three groups and grown to near confluence. Group 1 was incubated for 6 hours in 1% preshock or postshock lymph and ICAM-1 was measured by flow cytometry. Group 2 consisted of coculture of HMVECs and PMNs after endothelial cell activation to determine whether postshock lymph would stimulate PMN adherence. Group 3 was incubated under identical conditions, but PMNs were added for 30 minutes, and then activated with 4.5 micromol/L lysophosphatidylcholine (lyso-PC) for 1 hour to ascertain cytotoxicity. HMVEC density was measured using microscopy and recorded as HMVECs per millimeter squared. ICAM-1-blocking antibody and isotype control were used to assess the effects of ICAM-1 on PMN cytotoxicity. A buffer control was used for comparison using analysis of variance with Tukeys correction. RESULTS Postshock lymph activated HMVECs for increased surface expression of ICAM-1 and stimulated PMNs to adhere to endothelial cell monolayers. Activation of PMNs with lyso-PC in the presence of postshock lymph resulted in marked HMVEC death. The addition of an ICAM-1-blocking antibody abrogated this effect. Neither postshock lymph alone (758 +/- 35 HMVECs/mm(2)), nor postshock lymph in the presence of quiescent PMNs alone (734 +/- 28 HMVECs/mm(2)), nor lymph plus lyso-PC (834 +/- 21 HMVECs/mm(2)) provoked endothelial cell damage. CONCLUSION Postshock mesenteric lymph activates endothelial cells for increased ICAM-1 expression and PMN adherence. Furthermore, postshock lymph acts as an inciting event in a two-event in vitro model of PMN-mediated endothelial cell injury. These findings further substantiate the key mechanistic role of mesenteric lymph in hemorrhagic shock-induced acute lung injury and suggest that ICAM-1 expression is pivotal in the two-event model of multiple organ failure.

Hypertonic saline inhibits neutrophil (PMN) priming via attenuation of p38 MAPK signaling.

Priming of the neutrophil cytotoxic response is central to the pathogenesis of early postinjury multiple organ failure (MOF). Platelet-activating factor (PAF) has been implicated as a key inflammatory mediator in postinjury neutrophil priming and requires p38 MAPK signaling to produce its biologic effects. Hypertonic saline (HTS) resuscitation decreases the postinjury inflammatory response following shock in animals and decreases receptor-mediated neutrophil (PMN) cytotoxic functions in vitro. We hypothesized that HTS attenuates PAF priming of the PMN cytotoxic response by interfering with PAF-mediated p38 MAPK signal transduction. Isolated PMNs were preincubated in isotonic buffer or HTS (Na+ = 180 mM), then primed with PAF. Neutrophil CD11b/CD18 expression was measured by flow cytometry. Receptor-dependent (fMLP), N-formyl-methionyl-leucyl-phenylalanine, fMLP) and receptor-independent (PMA) O2- production was measured by reduction of cytochrome c in resting and PAF primed PMNs. Total p38 MAPK protein PAF-mediated p38 MAPK activation was assessed by western blot of PMN lysates. Clinically relevant levels of HTS attenuated PAF-mediated beta2-integrin expression. While HTS attenuated receptor-dependent (fMLP and PAF/fMLP) O2- production, receptor-independent (PMA) O2- production was unaffected. Conversely, HTS attenuated PAF priming of PMA-mediated O2- production. PAF and HTS did not alter total cellular p38 MAPK content. Clinically relevant levels of HTS alone did not activate p38 MAPK but inhibited PAF mediated p38 MAPK activation. HTS attenuates PAF priming of the PMN cytotoxic response by altering intracellular signal transduction. Therefore, HTS resuscitation may attenuate postinjury PMN priming and ultimately the risk of developing MOF.