Journal of Transplantation

Volume 2012 (2012), Article ID 793461, 9 pages

doi:10.1155/2012/793461

Review Article

Identification of Risk Factors for Vascular Thrombosis May Reduce Early Renal Graft Loss: A Review of Recent Literature

Anna Krarup Keller,1,2 Troels Munch Jorgensen,² and Bente Jespersen³

¹Institute of Clinical Medicine, Aarhus University, Denmark

²Department of Urology, Aarhus University Hospital, Brendstrupgaardsvej 100, Skejby, 8200 Aarhus N, Denmark

³Department of Nephrology, Aarhus University Hospital, Brendstrupgaardsvej 100, Skejby, 8200 Aarhus N, Denmark

Received 21 February 2012; Accepted 8 April 2012

Academic Editor: Gian Luigi Adani

Copyright © 2012 Anna Krarup Keller et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Renal graft survival has improved over the past years, mainly owing to better immunosuppression. Vascular thrombosis, though rare, therefore accounts for up to one third of early graft loss. We assess current literature on transplantation, identify thrombosis risk factors, and discuss means of avoiding thrombotic events and saving thrombosed grafts. The incidence of arterial thrombosis was reported to 0.2–7.5% and venous thrombosis 0.1–8.2%, with the highest incidence among children and infants, and the lowest in living donor reports. The most significant risk factors for developing thrombosis were donor-age below 6 or above 60 years, or recipient-age below 5-6 years, per- or postoperative hemodynamic instability, peritoneal dialysis, diabetic nephropathy, a history of thrombosis, deceased donor, or >24 hours cold ischemia. Multiple arteries were not a risk factor, and a right kidney graft was most often reported not to be. Given the thrombosed kidney graft is diagnosed in time, salvage is possible by urgent reoperation and thrombectomy. Despite meticulous attentions to reduce thrombotic risk factors, thrombosis cannot be entirely prevented and means to an early detection of this complication is desirable in order to save the kidneys through prompt reoperation. Microdialysis may be a new tool for this.

1. Background

In 1905, the first paper on kidney transplantation was published [1]. The French surgeon Alexis Carrel had, in the preceding years, developed techniques for vascular suture and anastomoses. In 1902, he transplanted a kidney from a small-size dog into its neck with the renal artery joined to the carotid artery and the vein to the external jugular vein. After three days, he noted a slightly greater blood flow than in the normal kidney and a urine production that was five times greater in the transplanted kidney. The animal died from an infection within few days. In 1912, Alexis Carrel received the Nobel Prize for his work.

Fifty years after the pioneering experiments, the first successful human renal transplantation was carried out in Boston by Dr. Joseph Murray in 1954 [2]. The donor was the patient’s identical twin brother. Preceding the operation, monozygosity was confirmed by the successful exchange of a full thickness skin graft between the brothers. The transplanted kidney functioned for eight years until cardiac death of the patient. In the succeeding decades, a tremendous development has been taking place, with an increasing understanding of immunological aspects of transplantation and the opportunity for pharmacological immunosuppression, which was revolutionized after introduction of cyclosporine in the late seventies. In 1990, Joseph E. Murray and E. Donnall Thomas received the Nobel Prize for their discoveries concerning organ and cell transplantation in the treatment of human disease.

In 2012, kidney transplantation remains the best treatment of end-stage renal disease (ESRD) resulting in improved health and quality of life [3] as well as decreased health expenses compared to sustained dialysis [4]. Renal graft survival has improved over the past years, mainly owing to better immunosuppression. As a consequence, although vascular thrombosis is a rare complication, it has become a major cause of early graft loss, accounting for up to one third of graft loss within one month [5] and up to 45–47% within two to three months [6]. In the pediatric NAPRTCS (North American Pediatric Renal Transplant Coorporative Study) cohort from 1996–2001, thrombosis was the most common cause of early graft loss [7].

2. Incidence of Vascular Thrombosis

The surgical technique has only seen minor changes since the first renal transplantations were accomplished. Usually the kidney is placed extraperitoneally in the iliac fossa with end-to-side anastomoses to the external iliac artery and vein. In small children and infants, however, this approach is often associated with an increased risk of thrombosis [8]. To avoid this, the kidney graft is anastomosed end-to-side to the distal abdominal aorta and caval vein.

Vascular thrombosis is a devastating complication because it most often leads to renal graft loss. Overall, the incidence of arterial thrombosis is reported to be 0.2–7.5% and venous thrombosis to be 0.1–8.2% as seen from Tables 1 and 2 with the highest incidence among children and infants and lowest in reports with only living donors. The incidence range is wide and many studies are not comparable due to differences in composition of recipients and donors, such as different percentage of deceased donors.

Table 1: RAT: Renal artery thrombosis, RVT: renal vein thrombosis, ¤: mean, *: % deceased, **: see text, c.s.: case series, c.r.: chart review, N/A: not available.

Author
Nationality
Year
N
Recipient age
Donor*
RAT + RVT
RAT
RVT
Study design

Jordan [9]
Canada, Toronto
1970–1980
341
13–67
84
4.4% (15)
3.5% (12)
0.9% (3)
Consecutive c.s.

Penny [5]
Australia, ANZDATA
1980–1992
6153
N/A
N/A
2.2% (134)
1.1% (70)
1.0% (64)
Case-control**

Murphy [10]
Ireland, Belfast
1989–1992
202
adults
N/A
4.5% (9)
1.0% (2)
3.5% (7)
Retrospective

Stechman [11]
UK, Oxford
1997–1992
401
27–75
78
1.0% (4)
0.75% (3)
0.25% (1)
Prospective

Benedetti [12]
USA, Minnesota
1985–1993
998
16–74
51
1.4% (14)
0.4% (4)
1.0% (10)
Retrospective

Bakir [6]
Holland, Groningen
1986–1994
558
14–71
100
6.1% (34)
2.0% (11)
3.4% (19)
Consecutive c.s.

Ojo [13]
USA, UNOS
1990–1996
84513
>18
N/A
0.9% (751)
N/A
N/A
Case-control**

Perez [14]
Spain, A Coruña
1988–1997
827
44¤
100
5.7% (47)
2.3% (19)
3.4% (28)
Retrospective

Englesbe [15]
USA, Michigan
1993–1997
714
all
43
1.82% (13)
0.8% (6)
1.0% (7)
Consecutive c.s.

Samhan [16]
Kuwait, Hawaly
1993–1998
151
all
24
3.3% (5)
2.6% (4)
0.7% (1)
N/A

Osman [17]
Egypt, Mansoura
1976–1999
1200
5–62
0
0.5% (12)
0.4% (5)
0.1% (1)
Consecutive c.s.

Mazzucchi [18]
Brazil, Sao Paulo
1995–1999
356
18–70
64
1.4% (5)
0.3% (1)
1.12% (4)
Retrospective c.r.

Parada [19]
Portugal, Coimbra
1980–2001
1000
41¤
98
0.9% (9)
0.6% (6)
0.3% (3)
Prospective

Orlic [20]
Croatia, Rijka
1971–2002
725
N/A
53
1.1% (8)
0.6% (4)
0.6% (4)
N/A

Hernández [21]
Spain, Tenerife
1996–2004
870
18–76
100
4.8% (42)
3% (26)
1.8% (16)
Retrospective c.r.

Sanni [22]
UK, Newcastle
1990–2005
1308
N/A
93
2.8% (36)
N/A
N/A
Retrospective

Dimitroulis [23]
Greece, Athens
1980–2005
1367
N/A
44
2.3% (31)
2% (27)
0.3% (7)
Consecutive

Salehipour [24]
Iran, Shiraz
1988–2006
1500
4–70
20
1.1% (16)
0.6% (9)
0.5% (7)
Retrospective c.r.

Amézquita [25]
Spain, Madrid
1990–2006
772
N/A
100
5.5% (42)
N/A
N/A
Case-control**

Table 2: Pediatric patients: RAT: Renal artery thrombosis, RVT: Renal vein thrombosis, *: % deceased, mc: multicenter, c.s.: case series, N/A: not available.

Author
Nationality
Year
N
Recipient age
Donor*
RAT + RVT
RAT
RVT
Study design

Harmon [26]
USA, NAPRTCS
1987–1989
1045
0–17
54
2.6% (27)
N/A
N/A
Prospective, mc.

van Lieburg [27]
Holland, Nijmegen
1977–1990
100
children
93
12% (12)
4% (4)
7% (7)
Retrospective

McEnery [28]
USA; NAPRTCS
1987–1992
2193
0–17
55
3.2% (71)
N/A
N/A
Prospective, mc.

Johnson [29]
UK, Ireland
1986–1995
1252
<18
100
4.0% (50)
N/A
N/A
Prospective, mc.

Ismail [30]
Poland, Warsaw
1984–1995
176
1–18
92
4.0% (7)
1.7% (3)
2.7% (4)
Retrospective

Kamel [31]
Ireland, Dublin
1986–1998
120
1–17
100
5% (6)
0.8% (1)
4.2% (5)
Prospective

Adams [8]
Germany, Heidelberg
1977–1998
61
1–6
77
13.1% (8)
4.9% (3)
8.2% (5)
Retrospective

Nagra [32]
UK, London
1987–2000
254
1–16
75
9.8% (25)
N/A
N/A
Retrospective

McDonald [7]
USA; NAPRTCS
1987–2001
7247
0–17
47
2.7% (199)
N/A
N/A
Prospective, mc.

Mickelson [33]
Canada, Vancouver
1984–2003
24
1–6
71
4.2% (1)
4.2% (1)
N/A
Retrospective

Kranz [34]
Germany, Essen
1998–2003
66
children
74
0
0
0
Prospective

El-Husseini [35]
Egypt, Mansoura
1976–2004
216
5–18
0
0
0
0
Consecutive c.s.

Garcia [36]
Brazil, Porto Alegre
1989–2005
40
1–5
25
10% (4)
7.5% (3)
2.5% (1)
N/A

In one of the studies with the lowest number of thrombosis [17], only living donors were used and furthermore the recipients were selected with an exclusion of diabetic patients. The pediatric patients from this cohort were later examined [35, 37] with no thromboses reported. This is unusual but could partly be due to selection of low risk donors between 21–60 years of age. In addition, there were no transplanted children below 5 years of age. In contrast, a subanalysis of NAPRTCS [26] revealed thrombosis-caused graft loss in 1.7% of all children receiving kidneys from a living donor. In the children less than 6 years of age, the incidence was 6.7%. These numbers are probably more reliable because NAPRTCS includes children of all ages and prospectively collected data on all pediatric patients in 73 centers [7].

Unfortunately, only few of the study data available are prospectively collected [7, 11, 19, 26, 28, 29, 31, 34], while most of them are retrospective. Five studies were named “consecutive case series” without thorough description of data collection [6, 9, 15, 17, 35]. This increases risk of bias and missing data. However, as thrombosis is an early complication that ultimately leads to graft loss in most cases, it is unlikely that it will go unnoticed. The question is of course, whether the cause of graft failure is correctly recorded in the medical chart. Three case-control studies are included in Table 1. Two of these included all cases from prospectively collected multicenter studies; Penny et al. [5] found 134 index cases of thrombosis in the Australian and New Zealand database, they selected an institutional control—which was the transplantation preceding the index case in the same institution and a donor graft control, the recipient of the kidney contralateral of the index case. Ojo et al. [13] identified 751 cases of thrombosis within the first 30 days in the prospectively collected United Network for Organ Sharing (UNOS) database, only 8 (1.1%) had to be excluded due to missing data. The cases were individually matched with two controls from the same year and the same transplantation center. The third case-control study is from a single center with the contralateral kidney as the only control [25]; the thrombosis case was solely included when the other kidney was transplanted in the same center thus leading to many exclusions and only 24 of 42 (57%) cases available for analysis.

3. Donors Risk Factors for the Development of Vascular Thrombosis

Historically, renal grafts with multiple arteries have been associated with an increased risk of thrombosis [38]; however, this does not seem to be the case in the modern transplant era [6, 12, 15, 17, 18, 21, 22, 27, 30, 32] where even a kidney with six arteries was successfully implanted [39]. Only one retrospective study from Iran reports significantly more surgical complications after multiple vessel kidney transplantation compared with recipients of single-artery graft [24]. In their statistical analyzes, they have analyzed hemorrhage, artery stenosis, renal artery thrombosis, and renal vein thrombosis all together, with the latter two being least frequent. It has previously been reported that multiple arteries is an independent risk factor for hemorrhage [17], ultimately leading to graft nephrectomy in 22% of the cases. This factor may carry weight in the first mentioned analysis, as they had a higher incidence of hemorrhage: 6.1% [24] compared to 1.9% in the latter [17]. No separate analysis for thromboses alone was provided from the Iranian center.

Surplus renal arteries are found in 10–20% of the population. In order to succeed in overcoming the technical challenges, it requires an experienced surgeon with an in-depth knowledge of possible variations in renal arterial anatomy and of microsurgical techniques for vascular reconstruction [39]. The arteries can either be anastomosed individually, in a Carrel patch (deceased donors), or they can be converted to a single artery, which will increase bench surgery time but decrease operative time for anastomosis.

There is no general consensus about whether a right renal graft has increased thrombosis risk compared to the left. A few centers, with both high and low thrombosis incidence, find no association [15, 17, 32], while others report a dramatic difference [6, 14, 25]. A recent publication from Spain [25] identified all kidney grafts thrombosed within 30 days after transplantation. The transplantation outcome of the contralateral kidney from the same donor was also identified and served as control. Twenty-four kidney pairs were analyzed and revealed no significant differences in recipients’ characteristics, but a huge difference in graft sides, with 21 thrombosed right kidney graft (87.5%) versus three left kidneys. This corresponds to the results from a center in The Netherlands [6], where 85% of the thrombosed kidneys were right versus 58% of the controls without thrombosis. The right kidney has a short vein and long artery, which can make it more difficult to position. The artery can easily be kinked and the vein is prone to compression by the kidney. This is supported by a significant association between right kidney and technical surgical problems in a multivariate analysis [6]. Like problems with multiple vessels can be overcome; right kidneys may not compose such a substantial risk factor, when operated by a meticulous surgeon.

The renal graft is often positioned in the contralateral iliac fossa, but also the same side as the original position can be used as there is no association between complications and the side of graft placement [6, 12, 15, 17].

On average, around 35% of all renal transplantations are carried out with a kidney from a living donor with large variation from country to country. When using a living donor, the anatomy is well known preoperatively and the procedure is scheduled. This often leads to better results and fewer thromboses [7], which is also evident from Tables 1 and 2. Many studies do not demonstrate an increased risk of thrombosis after receiving a kidney from a deceased donor compared to a living donor [12, 13, 15, 24, 30, 32]. This may be due to relatively small study populations with a different mix of donor compositions. However, in the NAPRTCS reports the overall thrombosis incidence has not declined in spite of a higher fraction of living donors in recent years [7, 26]. On the other hand, the question has been raised weather laparoscopic nephrectomy in fact comprises a risk factor. A large register study demonstrated significantly more frequent delayed graft function and acute rejection in children after laparoscopic compared to open living donor nephrectomy [40, 41]. However, this has been questioned in a recent publication from three large centers, where similar results for open and laparoscopic procedure have been found [42]. In both series, there have been a minimal number of thromboses and hence no statistical conclusions can be made.

A female donor doubles the risk of thrombosis in the case-control study from the Australian and New Zealand Dialysis and Transplant Registry [5], probably due to smaller vessel size, as suggested by the authors. However, this has not been confirmed in other studies [6, 13, 15, 27, 32].

Although few studies find no association between donor age and thrombosis [6, 12, 17], convincing studies point to the contrary [5, 7, 14, 15, 32]. There is no consensus on how the age analyzes should be performed, which may explain some of the dispute. In the former retrospective studies that found no association, one is analyzed for donor age >45 or <45 years [12], another used no donors from extreme age groups [17], and the last one did not divide into smaller age groups before analyzing and found no association within an adult donor population [6]. When stratified into age groups, a poorer outcome is found when the donors are very young [7, 14, 32], especially when less than six years of age [7, 14] or the recipient is a child. If the renal grafts are <7 cm in length, they are often transplanted en bloc into the recipient [43]. However, kidneys grow fast when transplanted into a larger recipient [43], and it may thus be desirable to transplant the kidneys individually whenever possible. Also donors >60 years increase the thrombosis risk [14], which might be connected with more atherosclerotic vessels (Table 3).

Table 3: Factors associated with increased risk of thrombosis.

Increased risk of thrombosis

Donors
Recipients

Donor age >60
Recipient age <5–6 or >50

Young donors <6 years
Peritoneal dialysis

Cold ischemic time >24 h
Recipient diabetes mellitus

Renal vessel atherosclerosis
Renal vessel atherosclerosis

Right kidney
History of thrombosis

Technical surgical problems

Illiac graft anastomosis, age <6 years

Hemodynamic instability

No use of aspirin

Delayed graft function

Warm ischemic time (WIT) has not been found to be a risk factor for thrombosis development [6, 13, 15, 17, 21]. This should be interpreted with caution, as it is very likely to be caused by similar low WIT for most kidneys. Cold ischemic time (CIT) varies more, and this is also reflected in the risk analyzes. Many studies state no increased risk with increasing CIT [6, 12, 13, 15, 21, 25, 30], often these reports have relatively low CIT at a mean of 20-21 hours [21, 25]. Other reports with fairly larger study populations state a higher thrombosis incidence with longer CIT [5, 7, 14, 32], especially when it exceeds 24 hours [7, 32]. This is based on univariate analyzes and little information is provided on the reasons for extended CIT, hence confounding events cannot be ruled out.

4. Recipient Risk Factors for Development of Vascular Thrombosis

As previously mentioned, the placement of the renal graft in small children may influence the thrombotic risk. In a retrospective German study, children aged 6 years or younger had kidney grafts transplanted either retroperitoneally to the iliac vessels in the fossa iliaca or extraperitoneally with end-to-side anastomoses to the distal aorta and caval vein [8]. The procedure was switched from the former to the latter in 1990, and when there was an equal number in both groups the outcomes were analyzed. Anastomoses to the iliac vessels strongly increased the risk of thrombosis, which occurred in 25.8% of the patients versus 3.3% of the children operated with aortic and caval anastomoses. In contrast, another study found no association between placement of the graft and loss due to thrombosis, but they included children up to 16 years of age and gave no separate analysis for the youngest children [32]. Although the aortic/caval approach is now accepted as the best for the small children, it is still debated whether trans- or extraperitoneal access is to be favored. Seemingly, extraperitoneal placement provides similar graft outcome, and it limits gastrointestinal complications considerably [44], hence this approach is used in most centers.

Intra- and postoperative hemodynamic instability is rarely mentioned in transplantation outcome reports, maybe because the data are not always accessible. Nevertheless, this may be an important contributor to thrombotic risk. In a retrospective report from The Netherlands, it was found to be an independent risk factor [6]. It was defined as period(s) of hypotension, mean arterial blood pressure ≤70 mmHg, for more than 10 minutes intraoperatively or during the first 48 hours postoperatively. This was the case among 32.5% of the patients who later suffered from acute vascular thrombosis, while only 4.5% of the controls had hypotensive period(s). 33.3% (11/33) of hemodynamic instable patients developed thrombosis and lost their graft. This has been described previously in another study, where one of twelve arterial thromboses was explained by intraoperative hypotension with visibly poor perfusion to the kidney [9]. A pediatric study suggests hemodynamic instability to be even more important in children and find the young child more prone to nontechnical thrombosis because of a relatively low cardiac output, particularly in relation to a much larger kidney that leads to less efficient renal perfusion [45], hence an aggressive fluid therapy and cardiovascular monitoring are crucial.

Due to transplantation being a relatively infrequent operation in many centers, some reports only have few patients of extreme ages and a difference was not always found [6, 12, 13, 17, 25]. In a NAPRTCS report with a substantial number of very young recipients down to 0-1 years of age together with older children, age was only approaching significance as a predictor of graft thrombosis [7]. In other pediatric reports, the patients developing a thrombosed graft are younger [27], usually below 5–6 years [26, 32]. Yet, excellent pediatric outcome with no thromboses is seen in some centers [46, 47], even though a high proportion of the children have been very young. In both reports, some of the success is attributed to a very aggressive fluid management, and they underscore the critical importance of appropriate fluid resuscitative measures in small recipients of adult-sized kidneys [47]. A minimum recipient weight of 5, preferable 10 kg is suggested prior to operation [36]. The operative technique is based on the weight and not the age of the child.

Also elderly recipients are more prone to graft thrombosis [48]. Nevertheless, it is becoming more common to transplant patients at older ages and hence, in 2007 up to 18.9% of transplantations in American centers were performed in patients >65 years. When the patients were appropriately selected, a short time graft survival similar to that of other adults was noted [49]. This was also the conclusion in another study, where they propose that chronological age barriers should be substituted by biological limits [50].

In the preceding paragraph, it was concluded that donor gender was of less importance. The same is the case for recipient gender, with only one large case-control study finding a 46% higher likelihood of thrombosis in women [13], while several others report no association [6, 7, 15, 17, 24, 25, 32]. Overall, 54–74% of reported recipients are male, in agreement with a higher prevalence of end-stage renal disease (ESRD) in men [51].

The underlying disease that causes ESRD in the patient also plays a role in the transplantation outcome. Of significance is diabetic nephropathy [6, 13, 15] with an increased thrombotic risk. This might be because of diabetic angiopathy, as also atherosclerosis of either the recipient or donor vessels increases the risk [21, 22]. However, a Spanish case-control study with rather few subjects in the analysis found no association with diabetes mellitus [25] and another found no connection to any underlying disease [5]. Systemic lupus erythematosus increases the thrombosis risk compared to hypertensive nephropathy [13]. This is most probable due to the antiphospholipid antibody syndrome, which has been pointed out earlier [52].

The progression of the renal disease and possible comorbidities determine when and how the patient should be treated. There are three renal replacement options when reaching ESRD: transplantation, hemodialysis (HD), and peritoneal dialysis (PD). The choice, order, and duration of treatment can have a substantial impact on transplantation outcome. Preemptive transplantation, that is, when the new kidney is transplanted before initiation of dialysis, while the native kidneys still have a high urine production, is preferred to avoid dialysis and associated especially cardiovascular risks, but it has been found to be associated with increased thrombosis risk compared to transplantation after initiation of HD [7, 13]. Furthermore, a high native urine production in combination with dialysis is a risk factor [27]. It is speculated that these patients may be more difficult to keep in a relatively volume-expanded state in the intra- and postoperatively. Preemptive transplantation is favored whenever possible, as its superior long-term graft survival is well established [53, 54]. However, more focus is warranted on aggressive and well-monitored fluid management of patients undergoing transplantation and preoperative stop of medical treatment like diuretics and antihypertensive drugs except for beta blockers.

Several studies with large patient cohorts find substantial more thrombotic graft loss among patients who have been on PD prior to transplantation compared to HD [7, 10, 13, 55], while just a few retrospective studies state the type of dialysis not to affect the graft outcome [6, 14]. An acquired thrombophilic state may be involved, possibly due to selective protein loss in the peritoneal fluid, like in nephrotic syndrome. Another contributing factor could be selection of patients; they might have been switched from HD to PD because of vascular access problems, which may indicate atherosclerosis. At least this switch has been found to increase risk more than PD in itself [13]. In a retrospective study, thrombosis has been reported to comprise up to 41% of early graft failure among PD patients and only 30% among HD patients [55]. Antithrombotic treatment of HD patients during sessions or a more efficient dialysis may contribute as well. However, these observations should be carefully interpreted, as the data for the cause of early graft loss were available from only one third of the cases in this study.

Inherited or acquired forms of thrombophilia increase the risk of thrombosis in general and hence also in a newly received renal graft and anticoagulation may be a sound consideration. Screening for thrombophilia has shown to decrease the number of thrombotic events [34, 56]. This can be done in different ways: one study has screened blood samples of all patients prior to transplantation [34], while another screened the patients by registration of history of prior thrombosis including deep venous thrombosis, multiple miscarriages, or family history of thrombosis among others [56]. Subsequently, these patients had thrombophilia confirmed by laboratory tests. Two patients not initially identified by this method developed thrombotic complications and had a hypercoagulable disorder documented afterward. Transplantation of recipients with nephrotic syndrome is a risk factor for thrombosis, particularly in small recipients where nephrectomy prior to transplantation should be considered.

Some studies are too underpowered to detect an effect of anticoagulation among unselected recipients. For instance, heparin is used prophylactic in case of vague risk factors [17], but it has also been shown not to affect the outcome [32]. In the latter study, only 254 patients were included, even though their power calculation required at least 1,500 patients in each arm. Furthermore, the patients were not randomized to heparin or not, but by 1994 heparin was implemented as routine treatment, and the patients were divided for analysis according to transplant year. Unfortunately, this kind of power calculation is rarely stated in transplantation reports, and few studies are prospective with randomization. Aspirin has been suggested to be a more convincing preventer of thrombosis [11, 57]. Nevertheless, this should be carefully administered as it increases the bleeding risk when graft biopsy is needed at short notice. To overcome this, low molecular weight heparin can be initiated perioperatively and used during the time when renal graft biopsy should be an option. Also administration of rabbit antithymocyte globulin in a pediatric population decreased the platelet count and reduced thrombosis risk significantly [31]. The distribution of thrombotic risk factors was similar in the groups, but it was not a randomized study.

Delayed graft function is strongly correlated to thrombosis and a vascular cause for decreased graft function should always be considered [6, 14, 21]. Neither the degree of HLA mismatch [5, 13, 25] nor immunosuppression with cyclosporine [5, 6, 26, 27] was correlated to thrombotic events.

5. Graft Salvage Is Possible

Diagnosis of vascular thrombosis can be time consuming, from request to implementation, and may thus be delayed. Consequently, kidney grafts developing thrombosis are most often lost, and the patient is returned to dialysis treatment. However, with early diagnosis and intervention, it is possible to save the kidney [16, 20, 33, 58–60]. Even up to 15–20 hours after onset of symptoms, salvage has been reported [61, 62]. Flow in the occluded vessel can be reestablished by open surgery with urgent thrombectomy [16, 33]. If the thrombosis is a late complication, local thrombolysis by an endovascular access [58, 59] may be tried; this is, however, most likely more risky in a recently operated patient with an increased risk of bleeding.

6. Methods for Diagnosis of Vascular Complications

Venous thrombosis classically presents itself with a swollen tender graft and hematuria, while arterial thrombosis can be more silent with a sudden drop in urine output. Intrarenal thrombosis may also be involved in severe rejections. Symptoms are often sparse, and the risk of delayed graft function is high. This can be masked by a prior low urine production or native kidney function in preemptive patients. Usually thrombotic events occur within the first 10 postoperative days, in 93% of the cases within day seven [5]. Arterial thrombosis is often presented later than venous thrombosis [14], but the latter can also occur as a late complication.

When vascular thrombosis is suspected clinically or in a routine screening, the diagnosis is confirmed by color Doppler flow examinations, which may also be used within the first hours after transplantation if the kidney is not functioning at once after revascularization. This technique detects vascular complications in general, including renal artery stenosis, with an overall sensitivity and specificity of 88% and 85%, respectively [63]. The accuracy of complete vascular thrombosis detection is higher but, nevertheless, false-positive as well as false-negative results can still be seen [64], and the diagnosis is often made too late to salvage the transplanted kidney, also due to delay from request to realization of the procedure. It is a static method that most often has to be repeated in patients with delayed graft function [65], because it is impossible to perform both ultrasound and biopsies at intervals frequent enough for safe early warnings, and these patients are at a higher risk of thrombosis, as mentioned above. When the thrombosed kidney is removed, it should be histopathologically examined to exclude rejection as the primary event [21]. During salvage surgery, a renal biopsy is often requested.

7. Reducing Early Renal Graft Loss

Despite meticulous attentions to the risk factors discussed, thrombosis is not completely preventable and means to a fast early detection of this complication are desirable in order to implement the above-mentioned opportunities for salvage. Some steps have been taken in this direction, and thermodiffusion has been suggested as a possible new dynamic diagnostic technique. A small thermodiffusion electrode placed in the tissue continuously measures the absolute microcirculation in mL/100 g tissue per minute. It has been applied to ischemic porcine kidneys [66] and livers [67]. Furthermore, it has been clinically applied to thirty renal transplant grafts [68]. All studies found a significantly lower microcirculation in the organs subjected to most ischemic damage, but this technique still needs further validation, and there have been no new publications on the subject since 2004.

Microdialysis is another minimally invasive technique for dynamic monitoring of the local metabolism, which is being increasingly used experimentally as well as in the clinic. A small catheter is placed in the tissue of interest and by simple diffusion through a semipermeable membrane; the concentration of local metabolites can be estimated. The reconstructive surgeons have used it for more than ten years for free flap monitoring, for example, when the esophagus is reconstructed by a free jejunal flap after carcinoma resection. Critical ischemia locally within the flap is detected by microdialysis; accordingly, the flap can be reoperated and revascularized in time [69]. The two-lumen catheter is continuously perfused and samples are taken every 30 or 60 minutes for analysis with results provided within minutes. The free flap is monitored for 4–7 days after surgery; this timeframe is also relevant following renal transplantation, where 93% of thromboses occur within the first 7 days [5]. Up till February 2012, more than 14,000 studies on microdialysis have been published; of these 183 have been made on kidneys including recent publications on renal ischemia. In pigs, microdialysis detects renal ischemia within 10–30 minutes, while metabolites remain at stable levels when no ischemia is present. This has been closely studied in different animal models of total ischemia [70–72], partial ischemia [73], autotransplantation with subsequent postoperative ischemia [74], or transplantation with risk of thrombosis [75]. All studies had equal conclusions, with a fast detection of renal ischemia by use of microdialysis.The first investigations on human kidneys undergoing nephrectomy due to cancer [76] have recently been carried out. They found similar results, indicating that a clinical application of the technique seems feasible.

8. Conclusions

Arterial and venous renal graft thromboses are rare, but nevertheless devastating complications that usually lead to graft loss and accounts for a considerable fraction of early graft losses. Especially in the situation with delayed graft function, there is a need for continuous monitoring to facilitate salvage and avoid explorative surgery when no thrombosis is present.

The most significant risk factors for thrombosis are donor-age below 6 or above 60 years or recipient-age below 5–6 years, per- or postoperatively hemodynamic instability, PD, diabetic nephropathy, a history of thrombosis, deceased donor, or more than 24 hours CIT. Multiple arteries and right kidney are not as severe risk factors as earlier thought, but should, nevertheless, be handled very carefully by the surgeon. A lot of the risk factors are not adjustable, and the kidneys are to be used as there is universal organ shortage and a need for kidneys even from marginal donors. When risk factors are present, the number of thrombotic events may be reduced, for example, by aggressive fluid management of pediatric patients or anticoagulation of thrombophilic patients. Furthermore, there is a need for early detection as thrombosis is not completely preventable. Microdialysis may be suitable for this.

Abbreviations

NAPRTCS: | North American Pediatric Renal Transplant Corporative Study
UNOS: | United Network for Organ Sharing
WIT: | Warm ischemic time
CIT: | Cold ischemia time
HD: | Hemodialysis
PD: | Peritoneal dialysis
TX: | Transplantation
ESRD: | End-stage renal disease.

References

A. Carrel and C. C. Guthrie, “Functions of a transplanted kidney,” Science, vol. 22, no. 563, p. 473, 1905.

J. M. Barry and J. E. Murray, “The first human renal transplants,” Journal of Urology, vol. 176, no. 3, pp. 888–890, 2006.

A. Yildirim, “The importance of patient satisfaction and health-related quality of life after renal transplantation,” Transplantation Proceedings, vol. 38, no. 9, pp. 2831–2834, 2006.

W. Greiner, K. Obermann, and J. M. Schulenburg, “Socio-economic evaluation of kidney transplantation in Germany,” Archives of Hellenic Medicine, vol. 18, no. 2, pp. 147–155, 2001.

M. J. Penny, B. J. Nankivell, A. P. Disney, K. Byth, and J. R. Chapman, “Renal graft thrombosis: a survey of 134 consecutive cases,” Transplantation, vol. 58, no. 5, pp. 565–569, 1994.

N. Bakir, W. J. Sluiter, R. J. Ploeg, W. J. van Son, and A. M. Tegzess, “Primary renal graft thrombosis,” Nephrology Dialysis Transplantation, vol. 11, no. 1, pp. 140–147, 1996.

R. A. McDonald, J. M. Smith, D. Stablein, and W. E. Harmon, “Pretransplant peritoneal dialysis and graft thrombosis following pediatric kidney transplantation: a NAPRTCS report,” Pediatric Transplantation, vol. 7, no. 3, pp. 204–208, 2003.

J. Adams, C. Güdemann, B. Tönshoff, O. Mehls, and M. Wiesel, “Renal transplantation in small children—a comparison between surgical procedures,” European Urology, vol. 40, no. 5, pp. 552–556, 2001.

M. L. Jordan, G. T. Cook, and C. J. Cardella, “Ten years of experience with vascular complications in renal transplantation,” Journal of Urology, vol. 128, no. 4, pp. 689–692, 1982.

B. G. Murphy, C. M. Hill, D. Middleton et al., “Increased renal allograft thrombosis in CAPD patients,” Nephrology Dialysis Transplantation, vol. 9, no. 8, pp. 1166–1169, 1994.

M. J. Stechman, N. Charlwood, D. W. Gray, and A. Handa, “Administration of 75 mg of aspirin daily for 28 days is sufficient prophylaxis against renal transplant vein thrombosis,” Phlebology, vol. 22, no. 2, pp. 83–85, 2007.

E. Benedetti, C. Troppmann, K. Giltingham et al., “Short- and long-term outcomes of kidney transplants with multiple renal arteries,” Annals of Surgery, vol. 221, no. 4, pp. 406–414, 1995.

A. O. Ojo, J. A. Hanson, R. A. Wolfe et al., “Dialysis modality and the risk of allograft thrombosis in adult renal transplant recipients,” Kidney International, vol. 55, no. 5, pp. 1952–1960, 1999.

F. M. Perez, A. Rodríguez-Carmona, F. T. Garcia, C. Tresancos, P. Bouza, and F. Valdés, “Peritoneal dialysis is not a risk factor for primary vascular graft thrombosis after renal transplantation,” Peritoneal Dialysis International, vol. 18, no. 3, pp. 311–316, 1998.

M. J. Englesbe, J. D. Punch, D. R. Armstrong, J. D. Arenas, R. S. Sung, and J. C. Magee, “Single-center study of technical graft loss in 714 consecutive renal transplants,” Transplantation, vol. 78, no. 4, pp. 623–626, 2004.

M. Samhan, T. Sinan, and M. Al-Mousawi, “Vascular complications in renal recipients,” Transplantation Proceedings, vol. 31, no. 8, pp. 3227–3228, 1999.

Y. Osman, A. Shokeir, B. Ali-el-dein et al., “Vascular complications after live donor renal transplantation: study of risk factors and effects on graft and patient survival,” Journal of Urology, vol. 169, no. 3, pp. 859–862, 2003.

E. Mazzucchi, A. A. Souza, W. C. Nahas, I. M. Antonopoulos, A. C. Piovesan, and S. Arap, “Surgical complications after renal transplantation in grafts with multiple arteries,” International Brazilian Journal of Urology, vol. 31, no. 2, pp. 125–130, 2005.

B. Parada, A. Figueiredo, A. Mota, and A. Furtado, “Surgical complications in 1000 renal transplants,” Transplantation Proceedings, vol. 35, no. 3, pp. 1085–1086, 2003.

P. Orlic, D. Vukas, I. Drescik et al., “Vascular complications after 725 kidney transplantations during 3 decades,” Transplantation Proceedings, vol. 35, no. 4, pp. 1381–1384, 2003.

D. Hernández, M. Rufino, S. Armas et al., “Retrospective analysis of surgical complications following cadaveric kidney transplantation in the modern transplant era,” Nephrology Dialysis Transplantation, vol. 21, no. 10, pp. 2908–2915, 2006.

A. Sanni, C. H. Wilson, H. Wyrley-Birch et al., “Donor risk factors for renal graft thrombosis,” Transplantation Proceedings, vol. 39, no. 1, pp. 138–139, 2007.

D. Dimitroulis, J. Bokos, G. Zavos et al., “Vascular complications in renal transplantation: a single-center experience in 1367 renal transplantations and review of the literature,” Transplantation Proceedings, vol. 41, no. 5, pp. 1609–1614, 2009.

M. Salehipour, H. Salahi, H. Jalaeian et al., “Vascular complications following 1500 consecutive living and cadaveric donor renal transplantations: a single center study,” Saudi Journal of Kidney Diseases and Transplantation, vol. 20, no. 4, pp. 570–572, 2009.

Y. Amézquita, C. Méndez, A. Fernández et al., “Risk factors for early renal graft thrombosis: a case-controlled study in grafts from the same donor,” Transplantation Proceedings, vol. 40, no. 9, pp. 2891–2893, 2008.

W. E. Harmon, D. Stablein, S. R. Alexander, and A. Tejani, “Graft thrombosis in pediatric renal transplant recipients. A report of the North American pediatric renal transplant cooperative study,” Transplantation, vol. 51, no. 2, pp. 406–412, 1991.

A. F. van Lieburg, M. C. de Jong, A. J. Hoitsma, F. G. M. Buskens, C. H. Schroder, and L. A. H. Monnens, “Renal transplant thrombosis in children,” Journal of Pediatric Surgery, vol. 30, no. 4, pp. 615–619, 1995.

P. T. McEnery, S. R. Alexander, K. Sullivan, and A. Tejani, “Renal transplantation in children and adolescents: the 1992 annual report of the north american pediatric renal transplant cooperative study,” Pediatric Nephrology, vol. 7, no. 6, pp. 711–720, 1993.

R. J. Johnson, S. Armstrong, M. A. Belger, et al., “The outcome of pediatric cadaveric renal transplantation in the UK and Eire,” Pediatric Transplantation, vol. 6, no. 5, pp. 367–377, 2002.

H. Ismail, P. Kaliciński, T. Drewniak et al., “Primary vascular thrombosis after renal transplantation in children,” Pediatric Transplantation, vol. 1, no. 1, pp. 43–47, 1997.

M. H. Kamel, P. Mohan, P. J. Conlon, D. M. Little, P. O'Kelly, and D. P. Hickey, “Rabbit antithymocyte globulin related decrease in platelet count reduced risk of pediatric renal transplant graft thrombosis,” Pediatric Transplantation, vol. 10, no. 7, pp. 816–821, 2006.

A. Nagra, R. S. Trompeter, O. N. Fernando et al., “The effect of heparin on graft thrombosis in pediatric renal allografts,” Pediatric Nephrology, vol. 19, no. 5, pp. 531–535, 2004.

J. J. Mickelson, A. E. MacNeily, J. Leblanc, C. White, and W. A. Gourlay, “Renal transplantation in children 15 Kg or less: the British columbia children's hospital experience,” Journal of Urology, vol. 176, no. 4, pp. 1797–1800, 2006.

B. Kranz, U. Vester, S. Nadalin, A. Paul, C. E. Broelsch, and P. F. Hoyer, “Outcome after kidney transplantation in children with thrombotic risk factors,” Pediatric Transplantation, vol. 10, no. 7, pp. 788–793, 2006.

A. A. El-Husseini, M. A. Foda, M. A. Bakr, A. A. Shokeir, M. A. Sobh, and M. A. Ghoneim, “Pediatric live-donor kidney transplantation in Mansoura Urology & Nephrology Center: a 28-year perspective,” Pediatric Nephrology, vol. 21, no. 10, pp. 1464–1470, 2006.

C. D. Garcia, V. B. Bittencourt, F. Pires et al., “Renal transplantation in children younger than 6 years old,” Transplantation Proceedings, vol. 39, no. 2, pp. 373–375, 2007.

A. A. Shokeir, Y. Osman, B. Ali-El-Dein, A. El-Husseini, M. El-Mekresh, and A. B. Shehab-El-Din, “Surgical complications in live-donor pediatric and adolescent renal transplantation: study of risk factors,” Pediatric Transplantation, vol. 9, no. 1, pp. 33–38, 2005.

L. Belli, L. de Carlis, L. S. Belli et al., “Thromboendoarterectomy (TEA) in the recipient as a major risk of arterial complication after kidney transplantation,” International Angiology, vol. 8, no. 4, pp. 206–209, 1989.

G. Orlando, G. Gravante, M. D'Angelo et al., “A renal graft with six arteries and double pelvis,” Transplant International, vol. 21, no. 6, pp. 609–611, 2008.

C. Troppmann, M. A. McBride, T. J. Baker, and R. V. Perez, “Laparoscopic live donor nephrectomy: a risk factor for delayed function and rejection in pediatric kidney recipients? a UNOS analysis,” American Journal of Transplantation, vol. 5, no. 1, pp. 175–182, 2005.

O. Salvatierra and M. Sarwal, “Vulnerability of small pediatric recipients to laparoscopic living donor kidneys,” American Journal of Transplantation, vol. 5, no. 2, pp. 201–202, 2005.

P. Chandak, N. Kessaris, A. Durkan, et al., “Is laparoscopic donation safe for paediatric recipients?—a study of 85 paediatric recipients comparing open and laparoscopic donor nephrectomy,” Nephrology Dialysis Transplantation, vol. 27, no. 2, pp. 845–849, 2012.

D. D. Nghiem, S. Hsia, and J. D. Schlosser, “Growth and function of en bloc infant kidney transplants: a preliminary study,” Journal of Urology, vol. 153, no. 2, pp. 326–329, 1995.

P. D. Furness III, J. B. Houston, S. A. Grampsas, F. M. Karrer, C. F. Firlit, and M. A. Koyle, “Extraperitoneal placement of renal allografts in children weighing less than 15 kg,” Journal of Urology, vol. 166, no. 3, pp. 1042–1045, 2001.

B. M. Churchill, C. A. Sheldon, G. A. McLorie, and G. S. Arbus, “Factors influencing patient and graft survival in 300 cadaveric pediatric renal transplants,” Journal of Urology, vol. 140, no. 5, pp. 1129–1133, 1988.

O. Ojogho, S. Sahney, D. Cutler et al., “Superior long-term results of renal transplantation in children under 5 years of age,” American Surgeon, vol. 68, no. 12, pp. 1115–1119, 2002.

O. Salvatierra, E. Alfrey, D. C. Tanney et al., “Superior outcomes in pediatric renal transplantation,” Archives of Surgery, vol. 132, no. 8, pp. 842–849, 1997.

N. S. Alakulppi, L. E. Kyllönen, J. Partanen, K. T. Salmela, and J. T. Laine, “Lack of association between thrombosis-associated and cytokine candidate gene polymorphisms and acute rejection or vascular complications after kidney transplantation,” Nephrology Dialysis Transplantation, vol. 23, no. 1, pp. 364–368, 2008.

A. Sener, E. J. Schweitzer, R. Munivenkatappa et al., “Deceased-donor renal transplantation in the geriatric population demonstrates equal graft survival compared with younger recipients,” Transplantation, vol. 87, no. 10, pp. 1549–1554, 2009.

G. P. Segoloni, M. Messina, R. Giraudi et al., “Renal transplantation in patients over 65 years of age: no more a contraindication but a growing indication,” Transplantation Proceedings, vol. 37, no. 2, pp. 721–725, 2005.

S. I. Hallan, J. Coresh, B. C. Astor et al., “International comparison of the relationship of chronic kidney disease prevalence and ESRD risk,” Journal of the American Society of Nephrology, vol. 17, no. 8, pp. 2275–2284, 2006.

S. Vaidya, C. C. Wang, C. Gugliuzza, and J. C. Fish, “Relative risk of post-transplant renal thrombosis in patients with antiphospholipid antibodies,” Clinical Transplantation, vol. 12, no. 5, pp. 439–444, 1998.

A. Mahmoud, M. H. Saïd, M. Dawahra et al., “Outcome of preemptive renal transplantation and pretransplantation dialysis in children,” Pediatric Nephrology, vol. 11, no. 5, pp. 537–541, 1997.

H. U. Meier-Kriesche and B. Kaplan, “Waiting time on dialysis as the strongest modifiable risk factor for renal transplant outcomes: a paired donor kidney analysis,” Transplantation, vol. 74, no. 10, pp. 1377–1381, 2002.

J. J. Snyder, B. L. Kasiske, D. T. Gilbertson, and A. J. Collins, “A comparison of transplant outcomes in peritoneal and hemodialysis patients,” Kidney International, vol. 62, no. 4, pp. 1423–1430, 2002.

P. E. Morrissey, P. J. Ramirez, R. Y. Gohh et al., “Management of thrombophilia in renal transplant patients,” American Journal of Transplantation, vol. 2, no. 9, pp. 872–876, 2002.

G. J. Murphy, R. Taha, D. C. Windmill, M. Metcalfe, and M. L. Nicholson, “Influence of aspirin on early allograft thrombosis and chronic allograft nephropathy following renal transplantation,” British Journal of Surgery, vol. 88, no. 2, pp. 261–266, 2001.

A. S. Chiu and D. N. Landsberg, “Successful treatment of acute transplant renal vein thrombosis with selective streptokinase infusion,” Transplantation Proceedings, vol. 23, no. 4, pp. 2297–2300, 1991.

J. G. Modrall, G. P. Teitelbaum, H. Diaz-Luna, and T. Berne, “Local thrombolysis in a renal allograft threatened by renal vein thrombosis,” Transplantation, vol. 56, no. 4, pp. 1011–1013, 1993.

T. Fathi, M. Samhan, A. Gawish, F. Donia, and M. Al-Mousawi, “Renal allograft venous thrombosis is salvageable,” Transplantation Proceedings, vol. 39, no. 4, pp. 1120–1121, 2007.

S. D. Clarke, J. A. Kennedy, J. C. Hewitt, J. McEvoy, M. G. McGeown, and S. D. Nelson, “Successful removal of thrombus from renal vein after renal transplantation,” British Medical Journal, vol. 1, no. 5689, pp. 154–155, 1970.

O. Rouviere, P. Berger, C. Beziat, et al., “Acute thrombosis of renal transplant artery: graft salvage by means of intra-arterial fibrinolysis,” Transplantation, vol. 73, no. 3, pp. 403–409, 2002.

N. Grenier, C. Douws, D. Morel et al., “Detection of vascular complications in renal allografts with color Doppler flow imaging,” Radiology, vol. 178, no. 1, pp. 217–223, 1991.

J. Gao, A. Ng, G. Shih et al., “Intrarenal color duplex ultrasonography: a window to vascular complications of renal transplants,” Journal of Ultrasound in Medicine, vol. 26, no. 10, pp. 1403–1418, 2007.

N. Perico, D. Cattaneo, M. H. Sayegh, and G. Remuzzi, “Delayed graft function in kidney transplantation,” The Lancet, vol. 364, no. 9447, pp. 1814–1827, 2004.

T. Kraus, E. Klar, B. R. Osswald et al., “Continuous measurement of porcine renal cortex microcirculation with enhanced thermal diffusion technology,” Journal of Surgical Research, vol. 61, no. 2, pp. 531–536, 1996.

A. Mehrabi, T. Kraus, G. Otto et al., “Quantification of hepatic microcirculation and intrahepatic shunt perfusion during experimental liver transplantation,” Transplantation Proceedings, vol. 30, no. 3, pp. 794–796, 1998.

M. Angelescu, T. Kraus, M. Wiesel, O. Hergesell, U. Haberkorn, and E. Klar, “Assessment of renal graft function by perioperative monitoring of cortical microcirculation in kidney transplantation,” Transplantation, vol. 75, no. 8, pp. 1190–1196, 2003.

H. B. Sorensen, “Free jejunal flaps can be monitored by use of microdialysis,” Journal of Reconstructive Microsurgery, vol. 24, no. 6, pp. 443–448, 2008.

K. J. Weld, C. Montiglio, A. C. Bush et al., “Predicting irreparable renal ischemic injury using a real-time marker in the porcine model,” Journal of Urology, vol. 180, no. 5, pp. 2218–2225, 2008.

K. J. Weld, C. Montiglio, A. C. Bush, H. H. Harroff, and R. D. Cespedes, “Real-time analysis of renal interstitial metabolites during induced renal ischemia,” Journal of Endourology, vol. 22, no. 3, pp. 571–574, 2008.

A. K. Keller, T. M. Jorgensen, L. H. Olsen, and L. B. Stolle, “Early detection of renal ischemia by in situ microdialysis: an experimental study,” Journal of Urology, vol. 179, no. 1, pp. 371–375, 2008.

A. K. Keller, T. M. Jorgensen, L. H. Olsen, and L. B. Stolle, “Detection of local metabolic changes after progressive and stepwise reduction of renal blood flow in pigs,” Transplantation Proceedings, vol. 41, no. 1, pp. 44–48, 2009.

A. K. Keller, T. M. Jorgensen, K. Ravlo, T. K. Nielsen, L. H. Olsen, and L. B. Stolle, “Microdialysis for detection of renal ischemia after experimental renal transplantation,” The Journal of urology, vol. 182, no. 4, pp. 1854–1859, 2009.

H. Fonouni, R. M. Tahmasbi, and M. Golriz, “Using microdialysis for early detection of vascular thrombosis after kidney transplantation in an experimental porcine model,” Nephrol Dial Transplant, vol. 27, no. 2, pp. 541–547, 2011.

K. J. Weld, K. Evearitt, P. Dixon, and R. D. Cespedes, “Effects of ischemia on human renal interstitial fluid metabolites,” Journal of Urology, vol. 181, no. 2, pp. 878–883, 2009.