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 �:	Transfusion of aged blood in head and neck free flap surgery: perioperative outcomes analysis
Scott Koepsell MD PhD1*, Mina Le MD2*, Amy Anne Lassig MD2, Bruce Lindgren MS3, Claudia Cohn MD PhD1, Samir S. Khariwala MD2
Division of Transfusion Medicine, Department of Laboratory Medicine and Pathology, University of Minnesota
Department of Otolaryngology � Head and Neck Surgery, University of Minnesota
Biostatistics and Bioinformatics Core, Masonic Cancer Center, University of Minnesota
*Drs. Koepsell and Le contributed equally to this work
Corresponding Author:

Samir S. Khariwala, MD
MMC 396
420 Delaware St SE
Minneapolis, MN, 55455
 HYPERLINK "mailto:Khari001@umn.edu" Khari001@umn.edu
Fax: 612 6252101
Tel: 612 625 9449

Word Count: 4088

Running Title: Transfusion of aged blood in free tissue transfer


Abstract
Background: Transfusion of old red blood cells (RBCs) occurs commonly at tertiary medical centers and is with decreased tissue oxygenation.  Patients who undergo microvascular free tissue reconstruction of the head and neck may be particularly sensitive to old RBCs because the anastomosis and microvasculature of the flap require smooth flow and sufficient blood flow and oxygenation, respectively.
Methods: A retrospective review of clinical and laboratory data in a series of consecutive cases of microvascular reconstruction receiving blood transfusions was performed at a tertiary academic medical center. All patients undergoing ablative surgery of the head and neck with free flap reconstruction between September 2007 and March 2011 were selected.  Rates of neck infection, wound dehiscence, free flap loss, need to return to the operating room for revision surgery, infection at the free flap donor site, and mortality were assessed as was age of RBCs transfused perioperatively or postoperatively.
Results: One hundred seven patients underwent head and neck surgery with microvascular flap reconstruction during the study period. Of these, seventy-seven patients received RBC transfusions (1 to 25 units) in the first five days of surgery. Statistical analysis revealed that transfusion of older blood was not associated with adverse perioperative outcomes in this patient population. Pre-operative anemia was associated with more post-operative transfusions, but not worse outcomes.
Conclusions: Within the confines of this retrospective study, the age of the RBC units transfused did not lead to increased morbidity following free tissue reconstruction in the head and neck. Still, the preponderance of evidence suggests that transfusion of aged blood should be minimized when possible in this population.  

Free tissue reconstruction has become the standard of care following ablation of advanced malignancies in the head and neck  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_1" \o "Wong, 2010 #76" 1]. A significant portion of complex head and neck reconstructive surgery is performed at tertiary medical centers, which often serve as a regional repository for donated blood that is nearing expiration  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_2" \o "Gusenoff, 2006 #77" 2]. As a result, transfusions given to those patients having surgery at academic centers commonly consist of aged blood. Data gathered from critical care patients suggest that the transfusion of aged blood results in a decrease in tissue oxygenation shortly following transfusion  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_3" \o "Kiraly, 2009 #80" 3]. It is reasonable to expect that this phenomenon may also occur in patients who have undergone free tissue reconstruction of the head and neck.
During storage, red blood cells (RBCs) undergo biochemical changes that may have deleterious effects when transfused into susceptible patients. For example, aged blood has increased free hemoglobin and microparticles that scavenge free nitric oxide, resulting in vasoconstriction and endothelial disruption after transfusion  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_4" \o "Donadee, 2011 #3" 4]. Microparticles that accumulate during RBC storage also appear to have potent activation effects on mediators of coagulation and inflammation  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_5" \o "Diamant, 2004 #10" 5,  HYPERLINK \l "_ENREF_6" \o "Jy, 2011 #13" 6]. Furthermore, significant reductions in RBC rheological performance and cell deformability have been observed in aged blood as compared to freshly collected blood  ADDIN EN.CITE <EndNote><Cite><Author>Berezina</Author><Year>2002</Year><RecNum>7</RecNum><DisplayText>[7]</DisplayText><record><rec-number>7</rec-number><foreign-keys><key app="EN" db-id="zsrwaax9uezrxjevv0zv95fpda9tsw2xdae9">7</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Berezina, T. L.</author><author>Zaets, S. B.</author><author>Morgan, C.</author><author>Spillert, C. R.</author><author>Kamiyama, M.</author><author>Spolarics, Z.</author><author>Deitch, E. A.</author><author>Machiedo, G. W.</author></authors></contributors><auth-address>Department of Surgery, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07103-2714, USA.</auth-address><titles><title>Influence of storage on red blood cell rheological properties</title><secondary-title>J Surg Res</secondary-title><alt-title>The Journal of surgical research</alt-title></titles><pages>6-12</pages><volume>102</volume><number>1</number><edition>2002/01/17</edition><keywords><keyword>Adenine</keyword><keyword>Adult</keyword><keyword>Blood Coagulation</keyword><keyword>Blood Donors</keyword><keyword>*Blood Preservation</keyword><keyword>Cell Size</keyword><keyword>Erythrocyte Deformability</keyword><keyword>Erythrocytes/*physiology</keyword><keyword>Female</keyword><keyword>*Hemorheology</keyword><keyword>Humans</keyword><keyword>Male</keyword><keyword>Microscopy, Electron, Scanning</keyword><keyword>Middle Aged</keyword><keyword>Sodium Chloride</keyword><keyword>Solutions</keyword><keyword>Time Factors</keyword><keyword>Ultrafiltration</keyword></keywords><dates><year>2002</year><pub-dates><date>Jan</date></pub-dates></dates><isbn>0022-4804 (Print)&#xD;0022-4804 (Linking)</isbn><accession-num>11792145</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/11792145</url></related-urls></urls><electronic-resource-num>10.1006/jsre.2001.6306</electronic-resource-num><language>eng</language></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_7" \o "Berezina, 2002 #7" 7]. Intriguingly, a recent study reported that transfusion of stored blood versus fresh blood increased the amount of non-transferrin-bound iron, which supported growth of a pathogenic strain of Escherichia coli  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_8" \o "Hod, 2011 #11" 8].
In addition to the biochemical evidence that longer-stored RBCs can harbor in vitro and in vivo storage defects, many retrospective studies have correlated transfusion of older blood with worse clinical outcomes, albeit with sometimes conflicting results  ADDIN EN.CITE <EndNote><Cite><Author>Lelubre</Author><Year>2009</Year><RecNum>14</RecNum><DisplayText>[9]</DisplayText><record><rec-number>14</rec-number><foreign-keys><key app="EN" db-id="zsrwaax9uezrxjevv0zv95fpda9tsw2xdae9">14</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Lelubre, C.</author><author>Piagnerelli, M.</author><author>Vincent, J. L.</author></authors></contributors><auth-address>Department of Intensive Care Medicine, Erasme University Hospital, Universite Libre de Bruxelles, Route de Lennik 808, Brussels, Belgium.</auth-address><titles><title>Association between duration of storage of transfused red blood cells and morbidity and mortality in adult patients: myth or reality?</title><secondary-title>Transfusion</secondary-title><alt-title>Transfusion</alt-title></titles><pages>1384-94</pages><volume>49</volume><number>7</number><edition>2009/05/21</edition><keywords><keyword>Adult</keyword><keyword>Blood Preservation/*adverse effects</keyword><keyword>Clinical Trials as Topic</keyword><keyword>Erythrocyte Transfusion/*mortality</keyword><keyword>Humans</keyword><keyword>Medline</keyword><keyword>Time Factors</keyword></keywords><dates><year>2009</year><pub-dates><date>Jul</date></pub-dates></dates><isbn>1537-2995 (Electronic)&#xD;0041-1132 (Linking)</isbn><accession-num>19453985</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/19453985</url></related-urls></urls><electronic-resource-num>10.1111/j.1537-2995.2009.02211.x</electronic-resource-num><language>eng</language></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_9" \o "Lelubre, 2009 #14" 9]. For example, retrospective studies on trauma patients have linked older blood with more adverse events  ADDIN EN.CITE <EndNote><Cite><Author>Leal-Noval</Author><Year>2008</Year><RecNum>15</RecNum><DisplayText>[10]</DisplayText><record><rec-number>15</rec-number><foreign-keys><key app="EN" db-id="zsrwaax9uezrxjevv0zv95fpda9tsw2xdae9">15</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Leal-Noval, S. R.</author><author>Munoz-Gomez, M.</author><author>Arellano-Orden, V.</author><author>Marin-Caballos, A.</author><author>Amaya-Villar, R.</author><author>Marin, A.</author><author>Puppo-Moreno, A.</author><author>Ferrandiz-Millon, C.</author><author>Flores-Cordero, J. M.</author><author>Murillo-Cabezas, F.</author></authors></contributors><auth-address>Neurotrauma Critical Care, Hospital Universitario Virgen del Rocio, Seville, Spain. sramon@cica.es</auth-address><titles><title>Impact of age of transfused blood on cerebral oxygenation in male patients with severe traumatic brain injury</title><secondary-title>Crit Care Med</secondary-title><alt-title>Critical care medicine</alt-title></titles><pages>1290-6</pages><volume>36</volume><number>4</number><edition>2008/04/02</edition><keywords><keyword>Adult</keyword><keyword>*Blood Preservation</keyword><keyword>Brain/*blood supply</keyword><keyword>Brain Injuries/physiopathology/*therapy</keyword><keyword>*Erythrocyte Transfusion</keyword><keyword>Humans</keyword><keyword>Male</keyword><keyword>Oxygen/*metabolism</keyword><keyword>Prospective Studies</keyword><keyword>Time Factors</keyword><keyword>Trauma Centers</keyword></keywords><dates><year>2008</year><pub-dates><date>Apr</date></pub-dates></dates><isbn>1530-0293 (Electronic)&#xD;0090-3493 (Linking)</isbn><accession-num>18379257</accession-num><work-type>Clinical Trial&#xD;Research Support, Non-U.S. Gov&apos;t</work-type><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/18379257</url></related-urls></urls><electronic-resource-num>10.1097/CCM.0b013e3181692dfc</electronic-resource-num><language>eng</language></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_10" \o "Leal-Noval, 2008 #15" 10], whereas prospective studies showed no significant differences in outcomes among patients receiving fresh or old blood  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_11" \o "Walsh, 2004 #17" 11]. Another example includes a well-publicized retrospective study that showed a higher mortality rate among cardiac surgery patients who received older blood  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_12" \o "Koch, 2008 #60" 12]. Despite many pitfalls in these studies, they helped serve as the impetus for several multicenter, randomized controlled trials that are currently underway to assess clinical outcomes related to the transfusion of fresh versus aged RBCs.
Regarding patients undergoing head and neck reconstructive surgery utilizing free flaps, interest has recently surged regarding the indications for, and outcomes associated with, blood transfusion  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_13" \o "Rossmiller, 2010 #81" 13-15]. To date, the effect of transfusing fresh versus old blood has not been studied in this patient population. This is of particular interest because the success of the free tissue transfers depends on microvascular anastomoses that may be exquisitely sensitive to RBC abnormalities. Specifically, the well-characterized reduction in RBC deformability seen in aged blood has the potential to compromise oxygen delivery within a flap�s newly revascularized microcirculation. Given that many patients undergoing head and neck reconstructive surgery require blood transfusions, we sought to examine the impact, if any, of age of transfused blood on perioperative outcomes.

Methods
This study was performed with the approval of the institutional review board at the University of Minnesota (IRB# 1103E97240). The hospital medical record database was searched for Current Procedural Terminology codes to identify 107 patients who underwent head and neck surgery with reconstruction utilizing a free tissue transfer between September 2007 and March 2011. Chart review was performed on all patients to identify the underlying clinical characteristics of the patient population and the following perioperative outcomes: postoperative infection, free flap or wound dehiscence, free flap loss (partial or total), need to return to the operating room for revision surgery, infection at the free flap donor site and mortality. Postoperative infection was defined as purulence expressed from the neck postoperatively. Dehiscence was defined as a separation of at least 1 cm of the suture line between the flap and native tissues or between the native skin edges of the neck incision.
Laboratory data on all of the patients were reviewed to identify all RBCs transfused, both during surgery and for the next four days following surgery, and the age of the RBC units at the time of transfusion.
The majority of patients in this retrospective sample received more than one transfusion prior to hospital discharge. For patients receiving multiple transfusions, the age of the RBC units was usually different between transfusions given to the same individual. Three parameters were considered to represent the impact that the age of the transfusions given to the same patient may have on clinical events: 1) the mean age across all units, 2) the maximum age across all units and 3) the minimum age across all units. Statistical analysis was performed on patients who received at least one blood transfusion and included all transfusions received during surgery as well as the four days following surgery. The occurrence of each perioperative outcome was compared to the mean, minimum and maximum age of the blood units transfused using the two-sample t-test. Additionally, a logistic regression model was used to evaluate these three RBC unit age parameters against the outcomes while adjusting for the number of units each patient received. The adjusted odds ratios and the 95% confidence intervals (CI) for these three parameters are reported. All statistical analyses were performed using SAS, version 9.3 (SAS Institute). P-values less than 0.05 were considered statistically significant.

Results
One hundred seven patients were identified during the study period. The demographics of the patient population are found in Table 1. Seventy-seven of the 107 patients received a transfusion within the first 5 days of surgery, with the number of RBC units transfused ranging from 0 to 25 (Figure 1). Eight of the 30 patients who did not receive any transfused RBCs (26.7%) had received preoperative radiotherapy. In contrast, 21 of the 77 who did receive transfusions (27.3%) had been treated with radiotherapy prior to surgery. Similarly, 5 of the 30 patients not receiving a RBC transfusion (16.7%) and 13 of the 77 patients receiving a RBC transfusion (16.9%) had received chemotherapy prior to surgery.  Using the Fisher�s exact test, neither preoperative radiation therapy nor chemotherapy was significantly associated with a greater likelihood of receiving a transfusion, with a p-value greater than 0.999 for both.
	Perioperative outcomes were assessed by chart review and compared to the mean, minimum or maximum age of the RBC units at the time of transfusion in 75 patients who received 11 or fewer RBC units within the first 5 days of surgery. The perioperative outcomes of neck infection, free flap dehiscence, free flap loss, the need to return to the operating room for revision surgery, infection at the free flap donor site and morbidity were not significantly related to any of the RBC unit age parameters (Table 2).  There was one case of flap loss and 5 patients who developed varying degrees of partial flap loss, all less than 50%.  The patient with complete flap loss was not included in the statistical analysis as no analysis could be performed with n=1 for this variable.  No significant trends between the age of transfused RBCs and outcomes were observed (Table 2).
Two patients were excluded from further analysis due to an unusually large number of transfusions of 15 and 25 units. Both of these patients had squamous cell carcinoma, previously treated with radiation and chemotherapy. The patient requiring 25 units of RBCs perioperatively developed flap congestion and underwent leech therapy, resulting in significant iatrogenic blood loss and subsequent transfusions. The patient requiring 15 units of RBCs perioperatively received multiple transfusions following the occurrence of multiple postoperative hematomas.
Because pre-operative anemia has been described to predict free flap failure  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_16" \o "Hill, 2012 #75" 16], the effect of pre-operative anemia, defined as a hemoglobin less than 10 g/dL, on outcomes was analyzed. No statistically significant difference was detected in patients with pre-operative anemia. However, patients with pre-operative anemia were more likely to receive more units of RBCs perioperatively (median = 4 units, range 1-11 units) compared to patients without preoperative anemia (median = 2 units, range 1-11 units) with a p-value of 0.012.
Discussion
	The principles of blood transfusion in surgical patients have undergone a shift over the last several years. Previously, clinicians often kept strict guidelines for transfusing patients, often with the goal of keeping hemoglobin concentrations above 10 g/dL. Our institution has currently instituted a strict policy of transfusion only when hemoglobin concentrations drop below 7 g/dL in patients who have no other overriding indications such as significant cardiac disease. Because of this policy, we suspect that, going forward, a smaller proportion (compared to 70.1% in this analysis) of our patients will receive blood transfusions. This policy stems in large part from acknowledgment of the fact that we, like many tertiary institutions, are most often transfusing aged blood to our patients. Kiraly et al compared critical care patients who received older blood (>21 days) with those receiving newer blood (<21 days) through near infrared spectroscopy monitoring of tissue oxygenation in the thenar eminence following transfusion  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_3" \o "Kiraly, 2009 #80" 3]. Their data demonstrated a dramatic decrease in tissue oxygenation of nearly 10% immediately following transfusion of aged blood. In contrast, those who received newer blood had evidence of increased tissue oxygenation.
	Furthermore, a recent study conducted by Rossmiller et al examined transfusion of patients following free tissue reconstruction in the head and neck by two separate criteria  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_13" \o "Rossmiller, 2010 #81" 13]. One group received transfusions for hematocrit less than 25, and the second group received transfusions for hematocrit less than 30. Each group contained approximately 133 free flap recipients and, upon analysis, the authors found a statistically significant decrease in postoperative respiratory failure and fistula formation in the group receiving transfusions only for hematocrit less than 25. In addition, the rate of free flap failure trended higher in the group receiving transfusion for hematocrit less than 30, although this did not reach significance. Given that their study was conducted at a tertiary institution, one can hypothesize that aged blood was given in most cases and therefore the trend towards increased flap failure could be related to greater loss of oxygenation in the larger number of patients receiving transfusions for hematocrit less than 30.
	Lastly, it is important to note that the literature does contain reports correlating transfusion with decreases in overall and disease-related survival. Although Szakmany et al suggest that blood-transfusion-related immunosuppression is responsible for these findings  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_15" \o "Szakmany, 2006 #82" 15]; these studies are somewhat difficult to interpret due to the issue of reverse causality. That is, it may be that those who have the most significant disease, and therefore worst outcomes, require more transfusions. Another report suggested that transfusion of three or more units of red blood cells in patients undergoing ablative surgery for oral cavity carcinoma was associated with worse survival  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_17" \o "Taniguchi, 2003 #78" 17]. However, not all patients in that study received free tissue transfers. To illustrate the conflicting data present in the literature regarding transfusions in this population, we note a third study by Fenner et al that divided patients undergoing free flap reconstruction into groups receiving greater than four, or less than four, units of red blood cell transfusions  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_18" \o "Fenner, 2009 #79" 18]. In that study, no differences in overall survival were seen between the two groups.
All retrospective studies suffer from limitations, and the existing literature describing the impact of the time of RBC storage is fraught with pitfalls such as not accounting for the number of units transfused. For example, patients with more morbidity are more likely to receive multiple transfusions and thus are more likely to receive an older unit of blood. This tendency will necessarily skew studies that report mean or maximum age of units transfused, especially when associated with worse outcomes  ADDIN EN.CITE <EndNote><Cite><Author>van de Watering</Author><Year>2011</Year><RecNum>68</RecNum><DisplayText>[19]</DisplayText><record><rec-number>68</rec-number><foreign-keys><key app="EN" db-id="zsrwaax9uezrxjevv0zv95fpda9tsw2xdae9">68</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>van de Watering, L.</author></authors></contributors><auth-address>Sanquin Blood Bank, Southwest Region, Leiden, The Netherlands. l.vandewatering@sanquin.nl</auth-address><titles><title>Pitfalls in the current published observational literature on the effects of red blood cell storage</title><secondary-title>Transfusion</secondary-title><alt-title>Transfusion</alt-title></titles><pages>1847-54</pages><volume>51</volume><number>8</number><edition>2011/08/13</edition><keywords><keyword>Bias (Epidemiology)</keyword><keyword>Blood Preservation/*adverse effects/*methods/standards/statistics &amp; numerical</keyword><keyword>data</keyword><keyword>Erythrocyte Transfusion/legislation &amp; jurisprudence/methods/standards</keyword><keyword>*Erythrocytes</keyword><keyword>Hospitalization</keyword><keyword>Humans</keyword><keyword>Observation</keyword><keyword>*Publication Bias</keyword><keyword>Publishing/standards/*statistics &amp; numerical data</keyword><keyword>Time Factors</keyword></keywords><dates><year>2011</year><pub-dates><date>Aug</date></pub-dates></dates><isbn>1537-2995 (Electronic)&#xD;0041-1132 (Linking)</isbn><accession-num>21831185</accession-num><work-type>Review</work-type><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/21831185</url></related-urls></urls><electronic-resource-num>10.1111/j.1537-2995.2010.03015.x</electronic-resource-num><language>eng</language></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_19" \o "van de Watering, 2011 #68" 19]. Despite this known pitfall, none of the analyzed perioperative complications in our study correlated with the age of the RBC units transfused either before or after we adjusted for the number of RBC units transfused (Table 2).
	Another potential limitation of this study is the number of patients studied. In the well-publicized report showing that older transfused blood was associated with higher morbidity and mortality among patients undergoing cardiac surgery, 6,002 patients were studied  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_12" \o "Koch, 2008 #60" 12]. The smaller number of patients analyzed in this study may still be valid because flap integrity is likely highly sensitive to the integrity of the microvascular anastomoses.
	Interestingly, we did not find an association between pre-operative anemia and worse outcomes (Table 3). This in contrast to a previous study that found flap failure increased when the patient�s pre-operative hemoglobin was less than 10 g/dL, with a relative risk of 4.76  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_16" \o "Hill, 2012 #75" 16]. This study evidenced a higher overall rate of flap loss (9%) and thus was more apt to identify associated factors.  Our study disputes this association albeit with significantly fewer flap failures.  These contrasting studies illustrate the difficulty in identifying factors associated with rare events.  
	Through analysis of our data, we have not identified negative outcomes in patients receiving aged blood transfusions during or following head and neck free flap reconstruction. Given that this patient population commonly requires transfusion within five days of surgery (70.1% in our study) and many require multiple transfusions (Figure 1), this finding is significant. Still, the biochemical and biomechanical changes that occur as blood ages are well documented.  As a result, physicians managing this complex group of patients should seek to eliminate unnecessary transfusion through the use of strict criteria while at the same time transfusing those patients who do require blood with the knowledge that doing so is unlikely to lead to additional perioperative complications.
Table 1
Patient sex
(n=107)46 female (43.0%)
61 male (57.0%)Pathology type
(n=107)86 squamous cell carcinoma (80.4%)
2 basal cell carcinoma (1.9%)
2 adenoid cystic carcinoma (1.9%)
2 sarcoma, not otherwise specified (1.9%)
1 myxofibroma (0.9%)
1 clear cell carcinoma (0.9%)
1 adenocarcinoma (0.9%)
1 leiomyosarcoma (0.9%)
1 dermatofibrosarcoma (0.9%)
1 hemangiopericytoma (0.9%)
1 angiosarcoma (0.9%)
1 odontogenic myxoma (0.9%)
1 myoepithelial carcinoma (0.9%)
1 malignant fibrous histiocytoma (0.9%)
1 Merkel cell carcinoma (0.9%)
1 invasive mucormycosis (0.9%)
1 Wegener�s granulomatosis (0.9%)
1 osteoradionecrosis (0.9%)
1 submucous fibrosis (0.9%)Pathology site
(n=107)69 oral cavity (64.5%)
13 cutaneous (12.1%)
9 sinonasal (8.4%)
8 oropharynx (7.5%)
3 hypopharynx (2.8%)
2 orbit (1.9%)
1 larynx (0.9%)
1 salivary gland (0.9%)
1 soft tissue (0.9%)Free flap donor site
(n=109)58 radial forearm (53.2%)
26 fibula (23.9%)
12 anterolateral thigh (11.0%)
9 rectus abdominis (8.3%)
2 scapula (1.8%)
2 latissimus dorsi (1.8%)
Table 2
10/3/12

Table 1: Comparing the mean, maximum and minimum age in days of the blood units received to outcomes following flap surgery.
OutcomeMean age of blood units (SD)p-value*Odds Ratio (95% CI)**Adjusted p-value�Max age of blood units (SD)p-value*Odds Ratio (95% CI)**Adjusted p-value�Min age of blood units (SD)p-value*Odds Ratio (95% CI)**Adjusted p-value�Flap infection
     No (n=56)
     Yes (n=19)
22.4 (9.4)
22.6 (8.3)0.9350.99
(0.73, 1.33)0.946
26.6 (10.7)
26.7 (  9.2)0.9901.00 
(0.78, 1.30)0.992
19.4 (9.7)
19.4 (8.8)0.9950.97
(0.71, 1.30)0.832Flap dehiscence
     No (n=56)
     Yes (n=18)
21.6 (9.1)
24.3 (8.7)0.2821.20
(0.88, 1.66)0.251
25.8 (10.5)
28.6 (  9.7)0.3241.14
(0.88, 1.50)0.320
18.9 (9.6)
20.1 (8.4)0.6391.09
(0.80, 1.48)0. 584Flap loss
     No (n=69)
     Partial (n=5)
     Complete (n=1) ��
22.5 (9.0)
19.1 (9.1)
35.9 (na)0.4230.84
(0.43, 1.49)0.577
26.8 (10.4)
22.8 (  9.6)
35.9 (na)0.4090.83
(0.50, 1.32)0.444
19.4 (9.4)
16.9 (9.2)
35.9 (na)0.5770.94
(0.48, 1.63)0.840Return to OR
     No (n=50)
     Yes (n=25)
23.0 (9.3)
21.4 (8.7)0.4670.92
(0.69, 1.22)0.553
27.1 (10.3)
25.7 (10.5)0.5760.94
(0.74, 1.19)0.581
20.2 (10.1)
17.8 (  7.9)0.3040.88
(0.65, 1.16)0.381Donor site infxn
     No (n=68)
     Yes (n=7)
22.2 (  9.0)
25.4 (10.1)0.3731.11
(0.72, 1.72)0.644
26.5 (10.4)
28.2 (10.3)0.6821.14
(0.76, 1.76)0.532
19.0 (  9.3)
23.1 (11.1)0.2811.08
(0.70, 1.64)0.724Donor site loss
     No (n=40)
     Yes (n=17)
22.1 (10.0)
22.9 (  6.9)0.7671.04
(0.75, 1.42)0.824
26.5 (11.7)
28.0 (  9.2)0.6361.09
(0.84, 1.45)0.520
19.0 (10.1)
19.2 (  7.2)0.9550.98
(0.70, 1.34)0.895Living on 10/30/11
     No (n=32)
     Yes (n=43)
22.4 (8.5)
22.5 (9.5)0.9680.98
(0.75, 1.28)0.866
26.9 (  9.7)
26.4 (10.8)0.8410.98
(0.78, 1.22)0.824
19.0 (9.2)
19.8 (9.7)0.7261.01
(0.78, 1.33)0.929* The p-value was obtained from the two-sample t-test.
� This p-value reflects the significance after adjusting for the number of units each person received using a logistic regression model.
** This is the odds ratio and confidence interval for a 5 day increase in the average mean/max/min of the age of the RBC units. 
�� The one patient with complete flap loss was excluded from the statistical analysis.
na is not applicable
Table 2

OutcomePercent with pre-op anemia
# (%)p-value*Flap infection
     No (n=56)
     Yes (n=19)
17 (30.4)
  3 (15.8)0.249Flap dehiscence
     No (n=56)
     Yes (n=18)
18 (32.1)
  2 (11.1)0.126Flap loss
     No (n=69)
     Partial (n=5)
     Complete (n=1) �
18 (26.1)
  2 (40.0)
  0 (  0.0)0.607Return to OR
     No (n=50)
     Yes (n=25)
14 (28.0)
  6 (24.0)0.788Donor site infxn
     No (n=68)
     Yes (n=7)
19 (27.9)
  1 (14.3)0.667Donor site loss
     No (n=40)
     Yes (n=17)
11 (27.5)
  3 (17.7)0.518Living on 10/30/11
     No (n=32)
     Yes (n=43)
11 (34.4)
  9 (20.9)0.291* The p-value was obtained from the Fisher�s exact test.
� The one patient with complete flap loss was excluded from the statistical analysis.
Figure 1
Figure legends
Table 1.Demographic data of the patient population including age, site of pathology, diagnosis, and flap donor site.
Table 2. Statistical analysis of the effect of mean, maximum and minimum age of the blood units received on outcomes following flap surgery.
Table 3 Statistical analysis of the effect of pre-operative anemia (hemoglobin <10.0 g/dL) on outcomes following flap surgery.
Figure 1. Histogram showing the distribution of the RBC units transfused during the surgery until the patient�s discharge from the hospital. Mean = 2.7 units, median = 2 units, mode = 0 RBC units.References
 ADDIN EN.REFLIST 1 Wong, C. H. & Wei, F. C. (2010) Microsurgical free flap in head and neck reconstruction. Head Neck 32: 1236-1245. doi:10.1002/hed.21284.
2 Gusenoff, J. A., Vega, S. J., Jiang, S., Behnam, A. B., Sbitany, H., Herrera, H. R., Smith, A. & Serletti, J. M. (2006) Free tissue transfer: comparison of outcomes between university hospitals and community hospitals. Plast Reconstr Surg 118: 671-675. doi:10.1097/01.prs.0000233203.84078.6b.
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4 Donadee, C., Raat, N. J., Kanias, T., Tejero, J., Lee, J. S., Kelley, E. E., Zhao, X., Liu, C., Reynolds, H., Azarov, I., Frizzell, S., Meyer, E. M., Donnenberg, A. D., Qu, L., Triulzi, D., Kim-Shapiro, D. B. & Gladwin, M. T. (2011) Nitric oxide scavenging by red blood cell microparticles and cell-free hemoglobin as a mechanism for the red cell storage lesion. Circulation 124: 465-476. doi:10.1161/CIRCULATIONAHA.110.008698.
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9 Lelubre, C., Piagnerelli, M. & Vincent, J. L. (2009) Association between duration of storage of transfused red blood cells and morbidity and mortality in adult patients: myth or reality? Transfusion 49: 1384-1394. doi:10.1111/j.1537-2995.2009.02211.x.
10 Leal-Noval, S. R., Munoz-Gomez, M., Arellano-Orden, V., Marin-Caballos, A., Amaya-Villar, R., Marin, A., Puppo-Moreno, A., Ferrandiz-Millon, C., Flores-Cordero, J. M. & Murillo-Cabezas, F. (2008) Impact of age of transfused blood on cerebral oxygenation in male patients with severe traumatic brain injury. Crit Care Med 36: 1290-1296. doi:10.1097/CCM.0b013e3181692dfc.
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14 Shah, M. D., Goldstein, D. P., McCluskey, S. A., Miles, B. A., Hofer, S., Brown, D. H., Irish, J. C., Gullane, P. J. & Gilbert, R. W. (2010) Blood transfusion prediction in patients undergoing major head and neck surgery with free-flap reconstruction. Arch Otolaryngol Head Neck Surg 136: 1199-1204. doi:10.1001/archoto.2010.202.
15 Szakmany, T., Dodd, M., Dempsey, G. A., Lowe, D., Brown, J. S., Vaughan, E. D. & Rogers, S. N. (2006) The influence of allogenic blood transfusion in patients having free-flap primary surgery for oral and oropharyngeal squamous cell carcinoma. Br J Cancer 94: 647-653. doi:10.1038/sj.bjc.6603013.
16 Hill, J. B., Patel, A., Del Corral, G. A., Sexton, K. W., Ehrenfeld, J. M., Guillamondegui, O. D. & Shack, R. B. (2012) Preoperative anemia predicts thrombosis and free flap failure in microvascular reconstruction. Ann Plast Surg 69: 364-367. doi:10.1097/SAP.0b013e31823ed606.
17 Taniguchi, Y. & Okura, M. (2003) Prognostic significance of perioperative blood transfusion in oral cavity squamous cell carcinoma. Head Neck 25: 931-936. doi:10.1002/hed.10313.
18 Fenner, M., Vairaktaris, E., Nkenke, E., Weisbach, V., Neukam, F. W. & Radespiel-Troger, M. (2009) Prognostic impact of blood transfusion in patients undergoing primary surgery and free-flap reconstruction for oral squamous cell carcinoma. Cancer 115: 1481-1488. doi:10.1002/cncr.24132.
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la�p�������ytZ�$$Ifa$gdZ�$d��$Ifa$gdB�d��$IfgdB��>�����������$���0������Ƅ��o�=�6�)���c����������������������������0�d��^��`�0�gdu	d�d�gd5n	d��gd�8�d��	d��gd1?������������DŽȄ…Åp�q�>�?�7�8�*�+�����e�f���������Z�[�����ӓԓ������–Ö���éÊp�p�p�p�p�p�p�p�p�p�p�p�p�p�p�p�p�p�p�2haYB*OJPJQJ^J_HmHnHph�tHu<hu	hu	B*OJPJQJ^J_HmHnHph�sHtHu3h?l�B*CJOJPJQJ_HmHnHph�sHtH<jh?l�B*CJOJPJQJU_HmHnHph�sHtH9h��h?l�B*OJPJQJ^J_HaJmHnHph�tH+����X���ѓ���������������������������������������������������������d��d�d�gdu	���0�d�^��`�0�gdu	���0�d��^��`�0�gdu	���������������������������������������������ū������������������h`l�#h`l�B*PJ_HmHnHph�tHh�A:jh�A:U3h�>�B*CJOJPJQJ_HmHnHph�sHtH<jh?l�B*CJOJPJQJU_HmHnHph�sHtH6hu	B*OJPJQJ^J_HmHnHph�sHtHu�����������������d�(��/ ��=!��"��#��$��%�������10:p(w��= ��/!��"��#��$��%�������.:p(w��/ ��=!��"��#��$��%�������ZD<EndNote><Cite><Author>Wong</Author><Year>2010</Year><RecNum>76</RecNum><DisplayText>[1]</DisplayText><record><rec-number>76</rec-number><foreign-keys><key app="EN" db-id="twvpfewaudxes6etrrkvxxwza0fd925frzft">76</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Wong, C. H.</author><author>Wei, F. 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