Microscopic Intrarenal Particles After Pulsatile Machine Preservation Do Not Adversely Affect Outcomes After Renal Transplantation

Microscopic Intrarenal Particles After Pulsatile Machine Preservation Do Not Adversely Affect Outcomes After Renal Transplantation J.V. Guarrera, S.H. Nasr, C.M. Reverte, B. Samstein, T. Brown, V. Balachandran, M.J. Samuels, J. Kelly, M.A. Hardy, G.S. Markowitz, V.D. D’Agati, and L.E. Ratner ABSTRACT Introduction. Our center has recently observed foreign carbohydrate-appearing particles (FP) on transplant postreperfusion biopsy specimens: (PRBx). Methods. To further characterize FPs, we reviewed all renal transplant RBx (30–45 minutes) performed between September 1, 2004 and December 3, 2005. Donor, preservation, and outcome variables were collected among patients with FP. Results. A total of 135 PRBx were performed (45 deceased donors [DD] and 90 live donors [LD]). Fifteen PRBx demonstrated FP. All 15 cases were DD kidneys that underwent machine perfusion (MP) on the Waters RM3 (Waters Medical Systems, Rochester, Minn, United States) with Belzer MP solution (Trans Med, Elk River, Minn, United States). Donor age was 39.8 ⫾ 15.7 years. Terminal creatinine level was 1.45 ⫾ 0.8 mg/dL. Two of 15 were flushed in situ with HTK solution (no starch). Cold ischemia time was 28.8 ⫾ 9.1 hours with 14.3 ⫾ 5.1 hours of MP. In 13 of 15 patients, perfusion parameters were excellent (flow ⬎ 100 mL; resistance ⬍ .35). Characteristics of FP. Particles were 10–30 ␮ and globular in shape. FP were not visible on hematoxylin and eosin stain, but stained strongly periodic acid-Schiff–(PAS) positive and were refractile under polarized light. FP were seen segmentally within glomerular capillaries in all cases and in peritubular capillaries in 3. In 11 of the 15 cases with FP, focal glomerular fibrin thrombi or intracapillary neutrophil margination was seen. Ten of 15 patients with FP had a biopsy within the first week with no identifiable FP. Outcomes. Recipient age was 45.3 ⫾ 11.6 years. Eight patients (53.3%) had delayed graft function. Biopsy-proven rejection occurred in 3 patients (20%). Three-month creatinine level was 1.59 ⫾ 0.35 mg/dL. One graft was lost to early thrombosis in a patient with a hypercoagulable state and 1 patient died of sepsis at 2 months. All remaining 13 patients are alive with excellent graft function at a median follow-up of 6.7 months (range, 3–17 months). Conclusions. Microscopic intrarenal particles may be seen on DD kidney PRBx after MP. These FPs likely originate from surgical gloves. FPs are too small to be captured by standard filters but clear spontaneously and do not have deleterious effects on renal function or outcomes.

A

STABLE rate of deceased donors1 and continued growth of the renal transplant waiting list has mandated the development of new sources of donor organs for

transplantation. Aggressive use of kidneys from extended criteria donors (ECD) has increased during the past decade. Hypothermic pulsatile machine perfusion (MP) and

From the Division of Abdominal Organ Transplantation (J.V.G., C.R., B.S., V.B., M.J.S., J.K., L.E.R.) and the Department of Pathology (S.H.N., G.S.M., V.D.D.), New York Presbyterian Hospital, New York, New York; and the Division of Transplantation, SUNY Downstate, Brooklyn, New York, USA.

Address reprint requests to James V. Guarrera, MD, Department of Surgery, New York Presbyterian Hospital, 525 East 68th Street Room M807, Box 20, New York, NY.

0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.10.168

© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710

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Transplantation Proceedings, 38, 3384 –3387 (2006)

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static cold storage (CS) have been the 2 available techniques for kidney preservation since the 1960s. Recently, there has been significant growth in MP with convincing registry data emerging that this technique increases use of ECD2 and in addition to improves early outcomes.3 Further improvements in preservation, pretransplantation assessment, and ex vivo resuscitation offer the means to achieve the full potential of this approach. Our program’s use of ECD kidneys has grown during the last several years in conjunction with a busy MP program in our region. Since September 2004, we have routinely performed 30- to 45-minute postreperfusion renal biopsies (PRBx). PRBx are an excellent baseline parenchymal evaluation that allows comparison of future allograft biopsy specimens. Since initiation of routine PRBx, we have observed microscopic foreign intrarenal carbohydrateappearing particles (FP). The source of these particles was initially thought to be Hydroxyethyl starch, which is an impermeant component of both University of Wisconsin Solution and the Belzer Machine Perfusion Solution. METHODS We retrospectively reviewed all renal transplant PRBx performed between September 1, 2004 and December 3, 2005. The aims were to identify and further characterize microscopic intrarenal FP. All renal biopsy samples were processed using standard techniques of light microscopy. For each case, we reviewed 11 glass slides stained with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), trichrome, and Jones methenamine silver. Donor, preservation, and outcome variables were collected for patients with FP.

RESULTS

Among 135 routine PRBx available for review included 45 from DD and 90 from live donors (LD). Fifteen PRBx demonstrated FP. All cases were DD kidneys that had been preserved with MP. None of the PRBx from LD showed FP (P ⬍ .0001).

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Fig 2. Foreign particles were also seen lodged in peritubular capillaries (arrow). (PAS, ⫻600.)

Histological Characteristics of FP

The mean number of glomeruli sampled for light microscopy was 11. The mean percentage of glomeruli with FP was 24% (range, 10%– 40%). The number of FP per glomerulus ranged from 1 to 4. FP were 10 to 30 ␮ and globular in shape. They were not visible on H&E and trichrome stains, but stained strongly PAS–positive (Fig 1). They were refractile (with birefringent crosses) under polarized light. FP were seen segmentally within glomerular capillaries in all cases and in peritubular capillaries in 3 cases (Fig 2). In 11 of the 15 cases with FP (73%), focal glomerular intracapillary fibrin-platelet thrombi or neutrophil margination was seen. Eight showed thrombi and neutrophil margination, 2 neutrophil margination only, and 1 thrombi only. In some cases, the fibrin-platelet thrombi were seen adherent to FP (Fig 3A and 3B). For comparison, glomerular intracapillary fibrin-platelet thrombi or neutrophil margination was seen in only 9 of the 30 (30%) DD transplant reperfusion biopsy specimens that did not show FP (P ⫽ .0062). Ten of 15 patients with FP (66%) had a repeat biopsy within 7 days of transplantation that did not show FP. Donor and Preservation Characteristics of Cases With Foreign Particles

Donor age was 39.8 ⫾ 15.7 years. Mean terminal creatinine level was 1.45 ⫾ 0.8 mg/dL. Total cold ischemia time was 28.8 ⫾ 9.1 hours. All patients underwent MP on the Waters RM3 (Waters Medical Systems, Rochester, Minn, United States) with Belzer Machine Perfusion Solution for at least 7 hours (mean, 14.3 ⫾ 5.1 hours). Perfusion parameters were excellent (flow ⬎ 100 mL; resistance ⬍0.35) in 13 of 15 kidneys. Two of the 15 grafts were flushed in situ with HTK solution, which does not contain hydroxyethyl starch. Patient Outcomes Fig 1. A representative glomerulus containing 3 PAS-positive globular intracapillary foreign particles (arrows). (PAS, ⫻600.)

The mean recipient age was 45.3 ⫾ 11.6 years. Eight patients (53.3%) had delayed graft function. Biopsy-proven

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Fig 3. (A) A glomerulus showing 2 intracapillary foreign particles (arrows), 1 of which is adherent to a fibrin-platelet thrombus (arrowhead). (PAS, ⫻400.) (B) The same glomerulus shown with the trichrome stain. The fibrin-platelet thrombus stains dark red (arrow). The starch particles are not apparent with this stain, (⫻400.)

rejection occurred in 3 patients (20%). One graft was lost to early thrombosis in a patient with a hypercoagulable disorder and 1 patient died of sepsis at 2 months. The remaining 13 patients are alive with excellent graft function at a median follow-up of 6.7 months (range, 3–17 months). Mean 3-month serum creatinine level was 1.59 ⫾ 0.35 mg/dL. DISCUSSION

Microscopic intrarenal particles may be seen on DD kidney PRBx. The issue of foreign particles being introduced into a transplanted organ during preservation is not new. The potential for adenosine crystal formation in defrosted UW solution has been demonstrated by Tullius et al.4 Currently, the UW package insert recommends using a 40-␮ filter during in situ flush. Although this filter would capture most foreign particles, it would not protect from the 10- to 30-␮ FP we have described in this report. We initially speculated that the source of the FP was

GUARRERA, NASR, REVERTE ET AL

HES, which is a component of both the Belzer UW and MP solutions. Two of the kidneys were flushed with HTK solution, which is devoid of HES, making it unlikely the UW flush solution is the culprit. All of the cases with FP were from DD and all had undergone MP, suggesting that Belzer MP solution or surgical gloves were the likely source. This observation also implied that if glove powder starch was the source, then it must require high pressure or recirculation that occurs during MP since none of the CS kidneys demonstrated FP. Several reports supported this hypothesis. Min et al.5 demonstrated similar particles in a dog kidney perfused continuously for 2 hours with Lactated Ringer’s solution containing starch glove powder. They further demonstrated that thorough washing of the gloves prevented particle deposition. In 1974, Elfenbein et al6 reported 2 transplants from the same donor with extensive starch particles, which were described as “emboli.” These kidneys were preserved using MP and the authors suspected extensive manipulation of the kidneys within the perfusion cassette as the source of the particles. One graft was lost early and the second at 7 weeks. These grafts showed persistence of particles in the explanted kidneys, which differs from our findings. In another report, Katz et al7 demonstrated that glove starch led to similar intrarenal particles in discarded human kidneys that underwent MP. These prior reports provided substantial evidence that surgical gloves are the culprit. The increasing use of “powder-free” gloves may be a factor in why only some of the MP kidneys have particles on PRBx. We found that the majority of PRBs with FP showed focal glomerular intracapillary fibrin-platelet thrombi and neutrophil margination; furthermore, in some biopsy specimens, the glomerular FP were surrounded by thrombi. This observation suggests that the FP may act as a nidus on which thrombi may form. Importantly, these focal thrombi in the setting of FP had no detectable deleterious effect on clinical outcome. Our report is the first transplant series of intrarenal starch particles in which outcome data is available. Two thirds of the patients had repeat biopsies with clearance of FP within 7 days of transplantation, and outcomes were similiar to most published series of DD renal transplants. In summary, our findings suggested that microscopic intrarenal starch particles were likely derived from starch powder in surgical gloves in conjunction with MP. The particles cleared spontaneously and did not have deleterious effects on renal function or outcomes.

REFERENCES 1. The Organ Procurement and Transplantation Network: Available at: http://www.optn.org. Accessed July 1, 2006 2. Schold JD, Kaplan B, Howard RJ, et al: Are we frozen in time? Analysis of the utilization and efficacy of pulsatile perfusion in renal transplantation. Am J Transplant 5:1681, 2005

MICROSCOPIC INTRARENAL PARTICLES 3. Matsuoka L, Shah T, Aswad S, et al: Pulsatile perfusion reduces the incidence of delayed graft function in expanded criteria donor kidney transplantation. Am J Transplant 6:1473, 2006 4. Tullius SG, Filatenkow A, Horch D, et al: Accumulation of crystal deposits in abdominal organs following perfusion with defrosted University of Wisconsin Solutions. Am J Transplant 2:627, 2002

3387 5. Min KW, Jackson JY, Gyorkey F, et al: Corn starch embolization in renal transplants. Kidney Int 2:291, 1972 6. Elfenbein IB, McAlack RF, Mills DCB, et al: Starch emboli in transplanted kidneys. Lancet 2:1009, 1974 7. Moriber-Katz S, Goldstein S, Ferluga D, et al: Contamination of perfused donor kidneys by starch from surgical gloves. Am J Clin Pathol 90:81, 1988