- Email: [email protected]
Diagnosis of renal allograft rejection and acute tubular necrosis by 99mTc-mononuclear leukocyte imaging
Diagnosis of Renal Allograft Rejection and Acute Tubular Necrosis by 99mTc-Mononuclear Leukocyte Imaging S.A. Lopes de Souza, L.M. Barbosa da Fonseca, R. Torres Gonçalves, D. Saloma˜o Pontes, T.J. Holzer, F.P. Proença Martins, and B. Gutfilen ABSTRACT One hundred kidney transplant recipients were evaluated on the first and fifth days after transplantation by Tc-99m mononuclear cell scintigraphy. We have developed a quantitative method to diagnose rejection and acute tubular necrosis (ATN) by comparing regions of interest drawn on allograft scintigraphs at different times after endovenous administration of the labeled cells. We suggest that the use of Tc-99m-WBC may be useful for the early diagnosis of rejection and the differential diagnosis of ATN.
T
RANSPLANTATION OF ORGANS has become increasingly successful. What was once an experimental and lifesaving emergency procedure has been transformed into a life-enhancing and technologically advanced form of therapy of chronic renal disease.1–3 The need to avoid unnecessary immunosuppressive therapy has led to a continued search for improved diagnostic methods for detection of rejection and its distinction from other complications.4 An acute rejection episode may occur during the first week posttransplantation. Although they may occur at any time, most rejections happen during the first 3 months. The cause is usually a T-cell–mediated immunological process. Clinically, acute rejection may be manifest by symptoms of fever, transplant tenderness, and kidney enlargement. Laboratory values, such as the erythrocyte sedimentation rate, serum creatinine, and 2-microglobulin levels, may rise.5 Another early complication that occurs, almost invariably with cadaveric but much less often with living related donor grafts, is acute tubular necrosis (ATN) due organ damage before transplantation. Since there is little if any destruction of tubular elements, the preferred current term is vasomotor nephropathy; the pathophysiological process is a reflex ischemic response within the kidney caused by local activation of the rennin-angiotensin axis.6 Many imaging methods have been used to evaluate the function and anatomy of renal allografts to diagnosis rejection. Among them are: renal arteriography, scintigraphy, ultrasonography, magnetic resonance imaging, and positron emission tomography. Despite the use of these methods, biopsy is still considered the gold standard to diagnose rejection.7–10
However, biopsy is an invasive diagnostic tool that can initiate graft damage in the first week posttransplantation. This study sought to develop a noninvasive diagnostic tool to diagnose early rejection and assist in the differential diagnosis of ATN. Since the rejection process is accompanied by the infiltration of lymphocytes, scintigraphy has been performed with In-111 or Tc-99m-labeled lymphocytes in animal models.11,12,13 To this end, we compared Tc-99m-mononuclear leukocyte scintigraphy at the first and fifth days posttransplantation with Doppler sonography, laboratory values, clinical features, and biopsy. We quantified changes in the signal in a region of interest (ROI) around the allograft area on the scintigram at specified time points.
MATERIALS AND METHODS One hundred consecutive renal transplant patients, 56 men and 44 women, engrafted from 89 related and 11 cadaveric donors were administered Tc-99m-mononuclear leukocytes followed by scintigraphy and Doppler ultrasonography. Allografts were obtained From the Departamento de Radiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. This work was financially supported by Fundação José Bonifácio/UFRJ (FUJB), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq). Address reprint requests to Prof Bianca Gutfilen, Universidade Federal do Rio de Janeiro, Hospital Universita´rio Clementino Fraga Filho, Departamento de Radiologia, Av. Brigadeiro Trompovsky s/n, Ilha do Funda˜o 21941-590, Rio de Janeiro, RJ, Brasil.
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.11.100
Transplantation Proceedings, 36, 2997–3001 (2004)
2997
2998
LOPES
following the transplantation sequence of the University Hospital. There were no exclusion criteria. The Institutional Review Board approved the study protocol, and informed written consent was obtained from all patients. Mononuclear leukocytes were labeled as previously described.14,15 Briefly, 30 mL venous blood was drawn into an aseptic vial, containing 1.0% of Liquemine (5000 Ul mL⫺1). The FicollHypaque technique for mononuclear cell isolation was followed according to English and Andersen.16 The mononuclear cells were washed by centrifugation in a saline solution using a clinical centrifuge. The pellet was resuspended in normal saline solution, placed in a Fisher tube and centrifuged for the removal of platelets (500 rpm for 1 minute at room temperature [RT]). This procedure was repeated three times to minimize the number of platelets when checked by microscopic observation. After the concentration of cells per milliliter was set at 107, stannous chloride (12 g/mL) was added for a 10-minute incubation at RT. After, 40 mCi (1480 MBq) of 99mTc was incubated for another 10 minutes, the supernate of the suspension was discarded, and the pellet resuspended. After another centrifugation, the final pellet was resuspended in saline for endovenous injection. Leukocytes were always labeled with freshly eluted Tc-99m. Labeling efficiency was always around 90% (range from 82% to 94%). Cell viability as determined using the trypan blue dye exclusion test was always greater than 95%. Scintigraphs were obtained using a Siemens Gamma Camera (Diacam, high-resolution, low-energy collimator, Germany) set for the gamma energy major peak of Tc-99m (140 keV) with a 15% window. Planar anterior views of the thorax and iliac fossa were obtained at 30 minutes and 3 and 24 hours after endovenous injection of approximately 12 mCi (444 MBq) of labeled cells. Each image was acquired for 5 minutes. Thorax images were obtained to check the normal biodistribution of the autologous Tc-99m-mononuclear leukocytes in the lungs, liver, and spleen. There was decreasing uptake over time of labeled cells in the lungs. The 24-hour images showed almost the entire injected dose localized in the target kidney allograft. Scintigraphs were performed on the first and fifth posttransplantation days; both data sets were used for the analysis. Doppler ultrasonography was obtained on the first and seventh posttransplantation days. We compared the first 10 patients’ scans with clinical and laboratorial findings. From this initial group of 10 patients, four patients were clinically rejecting at the fifth day as confirmed by biopsy. These images were compared with those of six patients whose clinical and laboratory findings were normal, in an attempt to find image alterations. Initially, it was difficult to detect a difference between the normal and the rejection images, since the renal allograft showed uptake of labeled cells in all patients. Thus, we decided to create an ROI on the scintigraphs for quantitative analysis of the 3- and 24-hour images. Rejecting allografts showed elevated uptake between the two images, which was quantitatively higher than that in patients who did not show evidence of rejection. A final dose of approximately 444MBq (12 mCi) of labeled cells was administered to each transplant patient. Allograft images on the first day, especially of cadaveric grafts, showed less activity in the 30-minute and 3-hour images (mean counts 20,000) than recipients with good clinical outcomes (mean counts 45,000) or patients with suspicion of rejection (mean counts 30,000). Thus, we decided to use the mean count percent differences between the 24- and 3-hour images and the total count of activity in the 30 minutes and 3-hour images to obtain a 99mTc-MNC diagnosis. We defined the diagnostic parameters as uptake ratio ⫽
DE
SOUZA, BARBOSA
DA
FONSECA, TORRES GONÇALVES ET AL
C24/C3 ⫻ 100 (C24: renal count rate at 24-hour postinjection; C3: renal count rate at 3-hour postinjection). The results were compared with the clinical analysis, which had the following characteristics high corporal temperature (above 37°C), high creatinine and urea levels (above 1, 2, and 40, respectively), physical exam of the graft, pain or enlargement and swelling of the graft, and diuresis (below 300 mL/h) and biopsy.
RESULTS
Clinical evaluation of the 100 renal transplant patients showed that during the first week posttransplant, 16 had rejection episodes in their allografts, five had a diagnosis of ATN, four had both ATN and rejection, and 79 had no abnormal features (Table 1). We observed the normal pattern to be a 10% to 15% count ratio between the 24- and 3-hour images, and total counts above 25,000 (Fig 1). Percentages above 15% were suggestive of rejection (Fig 2). The cutoff value was determined by a ROC curve (P ⬎ .05), as shown in Fig 3 and 4. The highest percentage measured by us was 36%. We also observed that all patients who had ATN showed total counts below 25,000 on the 3-hour images. Thus, we considered the value of 25,000 counts to be the cutoff for scintigraphies suggestive of ATN (Fig 5). The method was not able to differentiate acute rejection, which is associated with early ATN in cadaveric kidneys. All ATN cases were from cadaveric kidney donors. Five of the 16 rejection patients whose creatinine values decreased before day seven and increased after that day were correctly diagnosed at day 5 by the Tc-99m-MNC method. Patients that had both ATN and rejection were diagnosed at different days: ATN on the first day and rejection on the fifth day. The percentages of scintigraphic uptake are shown in Fig 6. Scintigraphy with 99mTc-mononuclear leukocytes enabled a correct diagnosis in 13 of the 16 rejections and four of the five ATN, while Doppler ultrasonography was diagnostic in 10 of 16 rejections and only two of five ATN. One of the 100 transplanted patients received an isogeneic graft. This individual was, surprisingly, the one with highest total count in the 3-hour image (148,429). Nevertheless the percentage of uptake was considered normal, correlating with the clinical, laboratory, and Doppler values and biopsy. One patient underwent dialysis during the interval of the 3- and 24-hour images. His percentage of uptake was above 15% even after the dialysis procedure, resulting in a diagnosis of rejection that was confirmed by biopsy. In this study, scintigraphy and ultrasonography showed Table 1. Outcomes of the Renal Transplant Recipients Patients (n)
Clinical outcome
16* 5* 79 Total 100*
Rejection ATN Successful
*Four patients had both ATN and rejection at the first week posttransplantation.
99MTC-MONONUCLEAR LEUKOCYTE IMAGING
2999
Fig 1. Normal uptake of Tc-99m-mononuclear leukocytes in a successful renal allograft.
sensitivities of 81% and 57% for rejection and 80% and 40% for ATN, respectively. There were no false-positive results for either method. Specificity was 100%. Accuracy was 96% and 91% for rejection and 99% and 97% for ATN, using scintigraphy and ultrasonography, respectively. The positive predictive value was of 100% for both methods. Negative predictive value was 95.1% and 89.7% for rejection and 98.9% and 96.9% for ATN, using scintigraphy and ultrasonography, respectively. The ROC curve P value was ⬎.005 and the confidential interval 0.780 to 0.988. Biopsies diagnosed rejection episodes in 16 patients and ATN in five. Tc-99m-MNC was used to diagnose 13 rejections and four with ATN for the same group of patients (Table 2).
Fig 3. ROC curve shows scintigraphic and histopathological diagnostic methods in renal transplantation.
was possible to predict a rejection episode early, then appropriate treatment may improve graft survival. Various approaches have been taken to address this question, such as examining the level of and presence of
DISCUSSION
The ability to detect early allograft rejection before advanced destruction remains a significant clinical goal. If it
Fig 2. Abnormal uptake of Tc-99m-mononuclear leukocytes in a renal allograft: rejection.
Fig 4. Percentage of uptake in the fifth day posttransplantation in patients with and without rejection.
3000
LOPES
Fig 5. Pattern of ATN: abnormal counts of Tc-99m-mononuclear leukocytes at 3-hour image.
activation markers on T-cell infiltrates, as well as the expression of inducible molecules, such as MHC class I, class II, and adhesion molecules.8 Although all of these parameters may indicate the presence of rejection, not one is a single reliable test. This study sought to diagnose early rejection, based on the fact that it is a T cell–mediated inflammatory response. In 100 renal transplant patients we labeled autologous mononuclear leukocytes with Tc-99m for infusion back into the patient for evaluation of their uptake into the allograft. Even the presence of mononuclear leukocytic interstitial nephritis did not guarantee the presence of acute rejection. During early rejection, acute cellular infiltrates into the interstitium and tubules can be a patchy, focal process, the functional status and clinical importance of which, unless clearly cytopathic, is not obvious.17
DE
SOUZA, BARBOSA
DA
FONSECA, TORRES GONÇALVES ET AL
Tc-99m-WBC and ln-111-WBC have been shown to diagnose heart and kidney allograft rejection in animal models,11–13 but to our knowledge the study presented here is the first to show the use of Tc-99m-WBC to evaluate human kidney allograft rejection. Among the 100 renal allograft transplantations, 16 experienced an acute rejection episode; four, both ATN and rejection; five, only ATN; and 79, without complications during the first week. Eight of the 79 patients with normal MNC-scintigraphy experienced rejection during follow-up after 30 days. There were three rejections led to death. Twenty-one patients underwent biopsies which confirmed 13 rejections and eight ATN. The fact that Tc-99mMNC correctly diagnosed five patients with decreasing creatinine values on the fifth and seventh days shows the capability of scintigraphy to detect early allograft rejection. In this study, we correctly diagnosed abnormal MNCscintigraphy that indicated the early presence of an acute rejection episode, around 2 to 3 days before clinical manifestations. We believe that this may be a valuable diagnostic tool to avoid unnecessary biopsies, mainly in patients with good laboratory findings and clinical evolution. Our observations in ATN may be explained by the fact that it is characterized by relatively good perfusion coupled with poor uptake and poor excretion.7 The low count of labeled mononuclear leukocytes could also be explained by the fact that ATN does not produce an infiltrate. The fact that dialysis did not interfere with the percentage uptake during rejection in one patient showed us this technique may be feasible in renal transplant patients. Doppler ultrasonography may be suggestive of acute rejection during the first week posttransplantation, but its sensitivity is low and its inability to distinguish rejection
Fig 6. Patients with percentage uptake higher than 15% were considered abnormal and suggestive of rejection.
99MTC-MONONUCLEAR LEUKOCYTE IMAGING
3001
Table 2. Comparison of Histopathology and Tc-99m-Mononuclear Leukocyte Scintigraphy Results Histopathology Scintigraphy
Positive
Negative
Positive Negative
17 4
0 79
from ATN has rendered it a less used method to evaluate immunological rejection. In addition, Hilborn et al reported that there was no statistically significant correlation of vascular grade with creatinine levels or resistive index in ATN versus rejection.18 Chows et al recently compared the Resistive Index (RI) and power Doppler readings in renal transplant patient biopsies. However, their results did not show a statistical significance among normal parenchymatous vascularization, RI, and rejection.19 The analysis of our results showed a PPV of 100% and a NPV of 95.1% for rejection and 98.9% for ATN. A positive predictive value of 100% implies that this method may be used to diagnose rejection with certainty and that treatment should be started immediately, which should increase the chances of saving the graft. We propose the use of 99m-Tc-mononuclear leukocytes for the follow-up of patients undergoing renal transplantation to allow early diagnosis of rejection and differential diagnosis of ATN.
REFERENCES 1. Allen RDM, Chapman JR: A manual of renal transplantation—British Library Cataloguing. London: Allen & Chapman; 1994 2. Ginns LC, Losimi AB, Morris PJ: Transplantation. 1st ed. Malden, Mass: Blackwell Science; 1999 3. Stites DP, Terr AI: Basic and clinical immunology. 7th ed. East Norwallk, Conn: Appleton & Lange; 1991
4. Thomsen HS: Renal transplant evaluation. In Murray IPC (ed): Nuclear medicine in Clinical Diagnosis and Treatment. New York: Churchill Livingstone; 1994, p 339 5. Thrail J, Ziessman HA: The Requisites—Nuclear Medicine. 2nd ed. St Louis, Miss: Mosby; 2001 6. Barros E, Manfro RC, Thomé FS, et al: Nefrologia—Rotinas, Diagnóstico e Tratamento. 2nd ed. Porto Alegre, Brazil: Artmed; 1999 7. Dunn EK: Radioisotopic evaluation of renal transplants. Urol Radiol 14:115, 1992 8. Heelan BT, Osman S, Blyth A, et al: Use of 2-[18F] fluoro2-deoxy glucose as potential agent in the prediction of graft rejection by positron emission tomography. Transplantation 66: 1101, 1998 9. Mostebeck GH, Reichhalter C, Stockenhuber F, et al: Comparison of Duplex sonography and color Doppler imaging in renal allograft evaluation: a prospective study. Eur J Radiol 10:201, 1990 10. Ye Q, Yang D, Williams M, et al: In vivo detection of acute rat renal allograft rejection by MRI with USPIO particles. Kidney Int 61:1124, 2002 11. Bergmann SR, Lerch RA, Carlson EM, et al: Detection of cardiac transplant rejection with radiolabeled lymphocytes. Circulation 65:591, 1982 12. Eisen HJ, Eisenberg SB, Saffitz JE, et al: Non-invasive detection of rejection of transplanted hearts with Indium-111labeled lymphocytes. Circulation 75:868, 1987 13. Farid NA, White SM, Heck LL, et al: Tc-99m labeled leukocytes: preparation and use in identification of abscess and tissue rejection. Radiology 148:827, 1983 14. Gutfilen B, Pontes LFS, Alencar ISB, et al: The development of a new and simple technique for labeling mononuclear cells with technetium 99m. Biomed Let 48:305, 1993 15. Gutfilen B, Pellini MP, Neder JR, et al: 99mTc-labeling white blood cells with a simple technique: clinical application. Ann Nucl Med 81:85, 1994 16. English D, Andersen BR: Single-step separation of red blood cells, granulocytes and mononuclear leukocytes on discontinuous density of Ficoll-Hypaque. J Immunol Methods 5:249, 1974 17. Pavlakis M, Lipman M, Strom TB: Intragraft expression of T-cell activation genes in human renal allograft rejection. Kidney Int 49(Suppl 53):S7, 1996 18. Hilborn MD, Bude RO, Murphy KJ, et al: Renal transplant evaluation with power Doppler sonography. Br J Radiol 70:39, 1997 19. Chow L, Sommer FG, Huang J, et al: Power Doppler imaging and resistance index measurement in the evaluation of acute renal transplant rejection. J Clin Ultrasound 29:483, 2001
T
RANSPLANTATION OF ORGANS has become increasingly successful. What was once an experimental and lifesaving emergency procedure has been transformed into a life-enhancing and technologically advanced form of therapy of chronic renal disease.1–3 The need to avoid unnecessary immunosuppressive therapy has led to a continued search for improved diagnostic methods for detection of rejection and its distinction from other complications.4 An acute rejection episode may occur during the first week posttransplantation. Although they may occur at any time, most rejections happen during the first 3 months. The cause is usually a T-cell–mediated immunological process. Clinically, acute rejection may be manifest by symptoms of fever, transplant tenderness, and kidney enlargement. Laboratory values, such as the erythrocyte sedimentation rate, serum creatinine, and 2-microglobulin levels, may rise.5 Another early complication that occurs, almost invariably with cadaveric but much less often with living related donor grafts, is acute tubular necrosis (ATN) due organ damage before transplantation. Since there is little if any destruction of tubular elements, the preferred current term is vasomotor nephropathy; the pathophysiological process is a reflex ischemic response within the kidney caused by local activation of the rennin-angiotensin axis.6 Many imaging methods have been used to evaluate the function and anatomy of renal allografts to diagnosis rejection. Among them are: renal arteriography, scintigraphy, ultrasonography, magnetic resonance imaging, and positron emission tomography. Despite the use of these methods, biopsy is still considered the gold standard to diagnose rejection.7–10
However, biopsy is an invasive diagnostic tool that can initiate graft damage in the first week posttransplantation. This study sought to develop a noninvasive diagnostic tool to diagnose early rejection and assist in the differential diagnosis of ATN. Since the rejection process is accompanied by the infiltration of lymphocytes, scintigraphy has been performed with In-111 or Tc-99m-labeled lymphocytes in animal models.11,12,13 To this end, we compared Tc-99m-mononuclear leukocyte scintigraphy at the first and fifth days posttransplantation with Doppler sonography, laboratory values, clinical features, and biopsy. We quantified changes in the signal in a region of interest (ROI) around the allograft area on the scintigram at specified time points.
MATERIALS AND METHODS One hundred consecutive renal transplant patients, 56 men and 44 women, engrafted from 89 related and 11 cadaveric donors were administered Tc-99m-mononuclear leukocytes followed by scintigraphy and Doppler ultrasonography. Allografts were obtained From the Departamento de Radiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. This work was financially supported by Fundação José Bonifácio/UFRJ (FUJB), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq). Address reprint requests to Prof Bianca Gutfilen, Universidade Federal do Rio de Janeiro, Hospital Universita´rio Clementino Fraga Filho, Departamento de Radiologia, Av. Brigadeiro Trompovsky s/n, Ilha do Funda˜o 21941-590, Rio de Janeiro, RJ, Brasil.
© 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.11.100
Transplantation Proceedings, 36, 2997–3001 (2004)
2997
2998
LOPES
following the transplantation sequence of the University Hospital. There were no exclusion criteria. The Institutional Review Board approved the study protocol, and informed written consent was obtained from all patients. Mononuclear leukocytes were labeled as previously described.14,15 Briefly, 30 mL venous blood was drawn into an aseptic vial, containing 1.0% of Liquemine (5000 Ul mL⫺1). The FicollHypaque technique for mononuclear cell isolation was followed according to English and Andersen.16 The mononuclear cells were washed by centrifugation in a saline solution using a clinical centrifuge. The pellet was resuspended in normal saline solution, placed in a Fisher tube and centrifuged for the removal of platelets (500 rpm for 1 minute at room temperature [RT]). This procedure was repeated three times to minimize the number of platelets when checked by microscopic observation. After the concentration of cells per milliliter was set at 107, stannous chloride (12 g/mL) was added for a 10-minute incubation at RT. After, 40 mCi (1480 MBq) of 99mTc was incubated for another 10 minutes, the supernate of the suspension was discarded, and the pellet resuspended. After another centrifugation, the final pellet was resuspended in saline for endovenous injection. Leukocytes were always labeled with freshly eluted Tc-99m. Labeling efficiency was always around 90% (range from 82% to 94%). Cell viability as determined using the trypan blue dye exclusion test was always greater than 95%. Scintigraphs were obtained using a Siemens Gamma Camera (Diacam, high-resolution, low-energy collimator, Germany) set for the gamma energy major peak of Tc-99m (140 keV) with a 15% window. Planar anterior views of the thorax and iliac fossa were obtained at 30 minutes and 3 and 24 hours after endovenous injection of approximately 12 mCi (444 MBq) of labeled cells. Each image was acquired for 5 minutes. Thorax images were obtained to check the normal biodistribution of the autologous Tc-99m-mononuclear leukocytes in the lungs, liver, and spleen. There was decreasing uptake over time of labeled cells in the lungs. The 24-hour images showed almost the entire injected dose localized in the target kidney allograft. Scintigraphs were performed on the first and fifth posttransplantation days; both data sets were used for the analysis. Doppler ultrasonography was obtained on the first and seventh posttransplantation days. We compared the first 10 patients’ scans with clinical and laboratorial findings. From this initial group of 10 patients, four patients were clinically rejecting at the fifth day as confirmed by biopsy. These images were compared with those of six patients whose clinical and laboratory findings were normal, in an attempt to find image alterations. Initially, it was difficult to detect a difference between the normal and the rejection images, since the renal allograft showed uptake of labeled cells in all patients. Thus, we decided to create an ROI on the scintigraphs for quantitative analysis of the 3- and 24-hour images. Rejecting allografts showed elevated uptake between the two images, which was quantitatively higher than that in patients who did not show evidence of rejection. A final dose of approximately 444MBq (12 mCi) of labeled cells was administered to each transplant patient. Allograft images on the first day, especially of cadaveric grafts, showed less activity in the 30-minute and 3-hour images (mean counts 20,000) than recipients with good clinical outcomes (mean counts 45,000) or patients with suspicion of rejection (mean counts 30,000). Thus, we decided to use the mean count percent differences between the 24- and 3-hour images and the total count of activity in the 30 minutes and 3-hour images to obtain a 99mTc-MNC diagnosis. We defined the diagnostic parameters as uptake ratio ⫽
DE
SOUZA, BARBOSA
DA
FONSECA, TORRES GONÇALVES ET AL
C24/C3 ⫻ 100 (C24: renal count rate at 24-hour postinjection; C3: renal count rate at 3-hour postinjection). The results were compared with the clinical analysis, which had the following characteristics high corporal temperature (above 37°C), high creatinine and urea levels (above 1, 2, and 40, respectively), physical exam of the graft, pain or enlargement and swelling of the graft, and diuresis (below 300 mL/h) and biopsy.
RESULTS
Clinical evaluation of the 100 renal transplant patients showed that during the first week posttransplant, 16 had rejection episodes in their allografts, five had a diagnosis of ATN, four had both ATN and rejection, and 79 had no abnormal features (Table 1). We observed the normal pattern to be a 10% to 15% count ratio between the 24- and 3-hour images, and total counts above 25,000 (Fig 1). Percentages above 15% were suggestive of rejection (Fig 2). The cutoff value was determined by a ROC curve (P ⬎ .05), as shown in Fig 3 and 4. The highest percentage measured by us was 36%. We also observed that all patients who had ATN showed total counts below 25,000 on the 3-hour images. Thus, we considered the value of 25,000 counts to be the cutoff for scintigraphies suggestive of ATN (Fig 5). The method was not able to differentiate acute rejection, which is associated with early ATN in cadaveric kidneys. All ATN cases were from cadaveric kidney donors. Five of the 16 rejection patients whose creatinine values decreased before day seven and increased after that day were correctly diagnosed at day 5 by the Tc-99m-MNC method. Patients that had both ATN and rejection were diagnosed at different days: ATN on the first day and rejection on the fifth day. The percentages of scintigraphic uptake are shown in Fig 6. Scintigraphy with 99mTc-mononuclear leukocytes enabled a correct diagnosis in 13 of the 16 rejections and four of the five ATN, while Doppler ultrasonography was diagnostic in 10 of 16 rejections and only two of five ATN. One of the 100 transplanted patients received an isogeneic graft. This individual was, surprisingly, the one with highest total count in the 3-hour image (148,429). Nevertheless the percentage of uptake was considered normal, correlating with the clinical, laboratory, and Doppler values and biopsy. One patient underwent dialysis during the interval of the 3- and 24-hour images. His percentage of uptake was above 15% even after the dialysis procedure, resulting in a diagnosis of rejection that was confirmed by biopsy. In this study, scintigraphy and ultrasonography showed Table 1. Outcomes of the Renal Transplant Recipients Patients (n)
Clinical outcome
16* 5* 79 Total 100*
Rejection ATN Successful
*Four patients had both ATN and rejection at the first week posttransplantation.
99MTC-MONONUCLEAR LEUKOCYTE IMAGING
2999
Fig 1. Normal uptake of Tc-99m-mononuclear leukocytes in a successful renal allograft.
sensitivities of 81% and 57% for rejection and 80% and 40% for ATN, respectively. There were no false-positive results for either method. Specificity was 100%. Accuracy was 96% and 91% for rejection and 99% and 97% for ATN, using scintigraphy and ultrasonography, respectively. The positive predictive value was of 100% for both methods. Negative predictive value was 95.1% and 89.7% for rejection and 98.9% and 96.9% for ATN, using scintigraphy and ultrasonography, respectively. The ROC curve P value was ⬎.005 and the confidential interval 0.780 to 0.988. Biopsies diagnosed rejection episodes in 16 patients and ATN in five. Tc-99m-MNC was used to diagnose 13 rejections and four with ATN for the same group of patients (Table 2).
Fig 3. ROC curve shows scintigraphic and histopathological diagnostic methods in renal transplantation.
was possible to predict a rejection episode early, then appropriate treatment may improve graft survival. Various approaches have been taken to address this question, such as examining the level of and presence of
DISCUSSION
The ability to detect early allograft rejection before advanced destruction remains a significant clinical goal. If it
Fig 2. Abnormal uptake of Tc-99m-mononuclear leukocytes in a renal allograft: rejection.
Fig 4. Percentage of uptake in the fifth day posttransplantation in patients with and without rejection.
3000
LOPES
Fig 5. Pattern of ATN: abnormal counts of Tc-99m-mononuclear leukocytes at 3-hour image.
activation markers on T-cell infiltrates, as well as the expression of inducible molecules, such as MHC class I, class II, and adhesion molecules.8 Although all of these parameters may indicate the presence of rejection, not one is a single reliable test. This study sought to diagnose early rejection, based on the fact that it is a T cell–mediated inflammatory response. In 100 renal transplant patients we labeled autologous mononuclear leukocytes with Tc-99m for infusion back into the patient for evaluation of their uptake into the allograft. Even the presence of mononuclear leukocytic interstitial nephritis did not guarantee the presence of acute rejection. During early rejection, acute cellular infiltrates into the interstitium and tubules can be a patchy, focal process, the functional status and clinical importance of which, unless clearly cytopathic, is not obvious.17
DE
SOUZA, BARBOSA
DA
FONSECA, TORRES GONÇALVES ET AL
Tc-99m-WBC and ln-111-WBC have been shown to diagnose heart and kidney allograft rejection in animal models,11–13 but to our knowledge the study presented here is the first to show the use of Tc-99m-WBC to evaluate human kidney allograft rejection. Among the 100 renal allograft transplantations, 16 experienced an acute rejection episode; four, both ATN and rejection; five, only ATN; and 79, without complications during the first week. Eight of the 79 patients with normal MNC-scintigraphy experienced rejection during follow-up after 30 days. There were three rejections led to death. Twenty-one patients underwent biopsies which confirmed 13 rejections and eight ATN. The fact that Tc-99mMNC correctly diagnosed five patients with decreasing creatinine values on the fifth and seventh days shows the capability of scintigraphy to detect early allograft rejection. In this study, we correctly diagnosed abnormal MNCscintigraphy that indicated the early presence of an acute rejection episode, around 2 to 3 days before clinical manifestations. We believe that this may be a valuable diagnostic tool to avoid unnecessary biopsies, mainly in patients with good laboratory findings and clinical evolution. Our observations in ATN may be explained by the fact that it is characterized by relatively good perfusion coupled with poor uptake and poor excretion.7 The low count of labeled mononuclear leukocytes could also be explained by the fact that ATN does not produce an infiltrate. The fact that dialysis did not interfere with the percentage uptake during rejection in one patient showed us this technique may be feasible in renal transplant patients. Doppler ultrasonography may be suggestive of acute rejection during the first week posttransplantation, but its sensitivity is low and its inability to distinguish rejection
Fig 6. Patients with percentage uptake higher than 15% were considered abnormal and suggestive of rejection.
99MTC-MONONUCLEAR LEUKOCYTE IMAGING
3001
Table 2. Comparison of Histopathology and Tc-99m-Mononuclear Leukocyte Scintigraphy Results Histopathology Scintigraphy
Positive
Negative
Positive Negative
17 4
0 79
from ATN has rendered it a less used method to evaluate immunological rejection. In addition, Hilborn et al reported that there was no statistically significant correlation of vascular grade with creatinine levels or resistive index in ATN versus rejection.18 Chows et al recently compared the Resistive Index (RI) and power Doppler readings in renal transplant patient biopsies. However, their results did not show a statistical significance among normal parenchymatous vascularization, RI, and rejection.19 The analysis of our results showed a PPV of 100% and a NPV of 95.1% for rejection and 98.9% for ATN. A positive predictive value of 100% implies that this method may be used to diagnose rejection with certainty and that treatment should be started immediately, which should increase the chances of saving the graft. We propose the use of 99m-Tc-mononuclear leukocytes for the follow-up of patients undergoing renal transplantation to allow early diagnosis of rejection and differential diagnosis of ATN.
REFERENCES 1. Allen RDM, Chapman JR: A manual of renal transplantation—British Library Cataloguing. London: Allen & Chapman; 1994 2. Ginns LC, Losimi AB, Morris PJ: Transplantation. 1st ed. Malden, Mass: Blackwell Science; 1999 3. Stites DP, Terr AI: Basic and clinical immunology. 7th ed. East Norwallk, Conn: Appleton & Lange; 1991
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