Although in the last 20 years, the 1-year immunological pancreas graft loss rate has decreased from 38 to 6% after pancreas transplantation alone (PTA), from 28 to 3.7% for pancreas after kidney transplantation (PAK), and from 7 to 1.8% after simultaneous pancreas–kidney transplantation (SPK), timely and accurate diagnosis of rejection of a pancreas graft is still essential and needs to be improved for solitary pancreas transplants. Whereas most graft losses because of acute rejection occur between months 7 and 12 after transplant, chronic rejection accounts for up to 30% of graft failure after the first year. According to the last International Pancreas Transplant Registry (IPTR) update, 75% of pancreases are transplanted together with a kidney, 18% following a kidney transplant, and 7% alone. Of the pancreas transplants performed outside the USA between 2000 and 2004, 91% were combined transplants, whereas in the USA, 71% of pancreas transplants performed during the same period were combined, 21% were PAK, and 8% PTA [1,2].
In the early days of pancreas transplantation, immunologic monitoring of the graft was based mainly on clinical symptoms, such as fever and pain as well as function and histology of the concomitantly transplanted kidney. Pancreatic duct drainage, be it after bladder or enteric drainage, permitted pancreatic enzymes in pure pancreatic juice to be precisely measured and led to the development of pancreatic juice cytology [3,4]. Urine amylase and lipase analysis following bladder drainage turned out to be a most helpful diagnostic tool. At that time, open or percutaneous biopsies were performed by only very few groups . The switch from bladder drainage to enteric drainage by the vast majority of pancreas transplant surgeons made it necessary to search for other methods of graft monitoring.
NONINVASIVE METHODS: BLOOD MARKERS
A number of assays have been established to detect markers that identify patients at higher risk for rejection including HLA antibodies, antiendothelial antibodies, the membrane glycoprotein CD 30, chemokines CXC ligand 9 and ligand 10, as well as determination of donor-specific memory T-cell reactivity [6▪]. These markers mainly describe the immune status of the recipient and have not been shown to be of great help in detecting acute rejection. Promising results have been reported by Cashion et al. , who used a real-time PCR assay of gene-expression levels of granzyme B, perforin, and HLA-DRA. Seven of thirteen patients with biopsy-proven rejection of their pancreatic allograft showed a significant increase in the level of these markers as early as 5 weeks before clinical rejection diagnosis. Even if the number of patients in this study is rather small, the results appear to confirm the larger experience with these two markers in kidney transplant recipients. The widely used measurement of amylase and lipase in the serum has been shown by Troxell et al.  to poorly correlate with biopsy-proven rejection: eight of 21 cases had normal serum studies.
NONINVASIVE METHODS: URINE BIOMARKERS
Some urine markers such as enzymes, neopterin, pancreas-specific protein, and insulin have been described to reliably indicate rejection in bladder-drained pancreatic grafts [9,10].
NONINVASIVE METHODS: PANCREATIC JUICE INVESTIGATIONS
Already in 1986, we reported on our early experience with monitoring of pancreatic allografts by the analysis of exocrine secretion. With this particular method, the quantity of pancreatic juice as well as its enzymatic and cellular content was evaluated . Pancreatic juice cytology was refined by our group and the Stockholm group  and became an extremely reliable tool for the detection of acute rejection. Later, we started to measure juice neopterin , which, however, turned out to be less specific than pancreatic juice cytology.
IMAGING TECHNIQUES: ULTRASONOGRAPHY
Already in 1996, Wong et al.  performed a study correlating the findings of gray scale and Doppler sonography with graft histology. In 36 patients clinically presenting with acute rejection, a sensitivity of only 58% was found. Doppler resistive index measurements in these patients turned out to widely overlap, irrespective of the severity of rejection.
In a more recent study, Gimenez et al.  concluded from 182 sonographic examinations in 51 consecutive pancreas transplant recipients that in the case of a vascular resistive index greater than 0.75 splenic vein thrombosis, pancreatitis or rejection should be suspected. If greater than 0.9, rejection must be seriously considered. Although ultrasound examination of the pancreatic allograft has been of little help in diagnosing acute pancreatic rejection, it is a valuable tool for monitoring graft perfusion .
IMAGING TECHNIQUES: MAGNETIC RESONANCE
Computed tomography (CT) is accurate in the assessment of extrapancreatic pathologies, but similar to ultrasound examination its unreliability in the detection of acute rejection has been demonstrated so far. In contrast, accuracy of contrast-enhanced magnetic resonance imaging has been proven by Krebs et al.  in 25 patients within 3 days of percutaneous biopsy or pancreatectomy (sensitivity 96%).
In very few patients, 111-indium-labeled autologous platelets were used for pancreas graft monitoring: five patients with an uneventful postoperative course showed normal uptake of the tracer in contrast to three patients, who lost their graft and showed an abnormal tracer uptake . The number of patients in whom this method has been applied is certainly too small to draw firm conclusions.
GRAFT BIOPSY: CYSTOSCOPIC TRANSDUODENAL
In the early 1990s, several groups began to perform transcystoscopic biopsies for the monitoring of bladder-drained pancreatic grafts. In this context, a cystoscopically directed needle biopsy technique under direct visualization was developed in a canine model . Nelson et al.  reported a success rate of 96%, even for ultrasonographically guided cystoscopic transduodenal biopsies. Macrohematuria in 4% and microhematuria in 31% were the most frequent complications associated with this technique .
GRAFT BIOPSY: LAPAROSCOPIC
Because of the lack of specific and sensitive rejection markers, mainly the Minneapolis group began to perform open biopsies in the late 1970s . This technique, however, has to a large extent been replaced by laparoscopic or more often percutaneous biopsy in enterically as well as bladder-drained grafts .
GRAFT BIOPSY: PERCUTANEOUS
After having established fine-needle aspiration in a canine model, the Sydney group was the first to report on their experience with percutaneous needle core biopsy of bladder-drained pancreatic grafts and thus comparing it with fine-needle aspiration cytology in humans. Their success rate for percutaneous needle core biopsy was reported at 93%, while fine-needle aspiration yielded sufficient diagnostic material in only 70% of patients . Transient hyperamylasemia occurred in 29% of the patients following core needle biopsy, but returned to baseline within 3 days. For guidance, some groups prefer ultrasonography and others CT scan, with similar success and complication rates between 2.8 and 11%. Complications include intra-abdominal hemorrhage, macrohematuria, allograft pancreatitis, exocrine leak, and inadvertent biopsy of other organs [22–25].
GRAFT BIOPSY: GASTROENTERIC DRAINAGE TECHNIQUES
In the era of enteric drainage, techniques were designed to permit direct endoscopic visualization of the pancreatic graft. Zibari et al.  described a technique in which the graft duodenum was anastomosed end-to-side to a Roux-en-Y loop, which was brought out as a temporary jejunostomy, thereby facilitating graft biopsy, collection of pancreas secretion, and pancreaticography under direct endoscopic vision. The same group performed a small series of pancreas transplants in which the end of the allograft jejunum was anastomosed to the anterior aspect of the stomach . The idea for gastric drainage of exocrine secretion goes back to Calne , who was the first to implant a pancreatic segment into the posterior wall of the stomach, a technique that was later followed by transplantation of the whole pancreas. The authors took biopsies from the duodenal portion of the graft in 28 instances without complications. One-year patient and graft survival of 94 and 85%, respectively, were reported.
Following the case report published by Hummel et al.  on a pancreas transplant with anastomosis of the graft duodenum to the lower third of the patient's duodenum, some other groups  adopted the same technique, which provides excellent access of the graft, but may create major problems when the native duodenum is closed after graft removal .
In order to avoid this risk, we began to take biopsies from the graft duodenum, which was hooked to the jejunum 25–50 cm beyond the ligament of Treitz. At that level, various endoscopic techniques such as push enteroscopy, single-balloon enteroscopy, or double-balloon enteroscopy seem to be similarly effective . With push enteroscopy, our group reached the graft duodenum in 76 out of 102 enteroscopies in a total of 65 pancreas recipients. Not even one intervention-related complication was observed. Apart from the fact that this investigation is successful in only two-thirds of all cases, the question arises whether biopsies from the graft duodenum are representative for the pancreatic portion .
Biglarnia et al.  recently presented the results of protocol biopsies of the graft duodenum which were obtained by double-balloon enteroscopy and classified according to the Banff criteria of small bowel allograft rejection. In 6 out of 29 patients, a rejection episode with normal pancreas function was detected and treated successfully. The authors conclude that protocol surveillance biopsies of the duodenal portion of the graft are effective in the early diagnosis of rejection.
CORRELATION BETWEEN REJECTION OF THE DUODENUM AND REJECTION OF THE PANCREAS
In a pig model, Sutherland and his group were the first to show complete concordance of rejection between the duodenum and the pancreatic parenchyma with regard to both interstitial and vascular findings in 15 (47%) of 32 animals. In 11 (65%) of the remaining 17 allografts, the pancreas was found to have a higher rejection rate and in six (35%) of the 17 the duodenum had a higher rejection rate. Every animal with histologically proven rejection of the pancreas also showed clear signs of rejection in the duodenum . In a rat model of pancreas, transplantation duodenal histology had a positive and negative predictive value of 100% for the detection of acute rejection in the pancreatic portion of the graft . When considering interstitial and vascular changes, vascular features correlated to a higher extent between the two portions of the graft than did interstitial features.
The Minneapolis group conducted an interesting study published in 1995 , in which they performed 52 cystoscopic duodenal biopsies, 25 of which were combined with a concurrent pancreas biopsy. Findings indicative for rejection in both organs agreed in nine cases (36%). In seven (28%) cases, rejection was diagnosed in both organs although the findings were discrepant with regard to the presence of vascular rejection (six pancreases and one duodenum). Minor nonrejection discrepant findings were present in another two (11%) cases. Thus, in 18 (72%) of 25 pancreaticoduodenal biopsies, a different therapy would not have been indicated if only one graft had been biopsied. The authors concluded that the duodenum and the pancreas can reject independently of each other, and a negative biopsy does not preclude rejection of the other organ. Furthermore, tissue samples of one organ alone are sufficient only in the case of positive findings.
Another group performed 53 mucosal biopsies of the pancreas and the kidney together in bladder-drained transplants. Pancreas biopsies were performed in the case of clinically suspected rejection (decreased urinary amylase, increased serum amylase, or increased serum creatinine) or by protocol. The authors report on postinterventional bleeding in 8% and hyperamylasemia in 20% of cases. Of the 14 duodenal biopsies revealing histology consistent with rejection, 14 correlated with pancreas rejection. Not even one of the negative duodenal mucosa specimens showed evidence of rejection in the absence of pancreatic rejection . In contrast, Carpenter et al.  obtained 17 pancreatic and 14 duodenal biopsies from allografts with exocrine drainage into the bladder. The histologic changes in the duodenum paralleled those in the pancreas in both rejecting and nonrejecting allografts.
Again, it was the Minneapolis group that attempted to clarify the question whether the duodenum serves as a marker for kidney rejection. If duodenal biopsies in that experimental study were consistent with rejection, they were highly representative for kidney rejection. A negative duodenal biopsy, however, did not rule out rejection of the kidney .
THE KIDNEY AS SURROGATE MARKER FOR THE PANCREAS
Only one group recently published their experience with 21 concurrent biopsies of the pancreas and the kidney after combined transplantation. Thirteen pairs (62%) were found to be concordant for rejection. Eight pairs (38%) were discordant: six showed rejection of the pancreas but not of the kidney and two pairs showed rejection of the kidney but not of the pancreas .
As all markers tested and also various imaging techniques have proven to have poor reliability with regard to diagnosing pancreatic allograft rejection, graft histology remains the only useful tool for this purpose. The duodenal portion of the pancreaticoduodenal graft is easily accessible in bladder-drained grafts and may be reached endoscopically in a high percentage of enterically drained grafts. In this context, duodenal histology appears to be a good surrogate marker for pancreatic rejection.
Conflicts of interest
There are no conflicts of interest to declare.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 123).
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