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ÍNDICE
Introducción
7
M. Arias
Early diagnosis of chronic allograft nephropathy
by means of protocol biopsies
13
J. Chapman
Diagnóstico precoz del rechazo subclínico
mediante biopsias de protocolo
31
F. J. Moreso
Protocol biopsies and the diagnosis of humoral rejection
51
M. Mengel
Adaptación glomerular evaluada mediante biopsias
de protocolo
71
Biopsia de protocolo: aspectos técnicos y seguridad
91
R. Palomar
Biopsias de protocolo en el diseño de ensayos clínicos
105
D. Serón
4
ÍNDICE DE AUTORES
COORDINADOR GENERAL
Manuel Arias Rodríguez
Jefe de Servicio y Catedrático de Nefrología
Hospital Universitario Marqués de Valdecilla (Santander)
COORDINADORES DEL VOLUMEN V
Manuel Arias Rodríguez
Jefe de Servicio y Catedrático de Nefrología
Hospital Universitario Marqués de Valdecilla (Santander)
Daniel Serón Micas Servicio de Nefrología
Hospital Universitari de Bellvitge (L’Hospitalet de Llobregat, Barcelona)
AUTORES
Jeremy Chapman OAM, MD, FRACP, FRCP Centre for Transplant and Renal Research Millennium Institute, University of Sydney
Westmead Hospital, Westmead (New South Wales, Australia) Xavier Fulladosa Oliveras
Servicio de Nefrología
Hospital Universitari de Bellvitge (L’Hospitalet de Llobregat, Barcelona) Michael Mengel
Institute for Pathology
Francesc Josep Moreso Mateos Servicio de Nefrología
Hospital Universitari de Bellvitge (L’Hospitalet de Llobregat, Barcelona) Rosa Palomar Fontanet
Servicio de Nefrología
Hospital Universitario Marqués de Valdecilla (Santander)
6
BIOPSIA DE PROTOCOLO
EN EL TRASPLANTE RENAL:
¿HERRAMIENTA CLÍNICA
O DE INVESTIGACIÓN?
M. Arias
El seguimiento clínico del trasplante renal se basa fundamentalmente en el análisis de la creatinina sérica y/o del filtrado glomerular generalmente por estimación mediante fórmulas. Se sabe que este método diagnóstico es insuficiente y que no se correla-ciona bien con la situación real del injerto. La otra opción posible es la realización de biopsias de protocolo en pacientes estables y asintomáticos, mientras no se desarro-llen test de monitorización inmunológica fáciles de efectuar, reproducibles y válidos para el diagnóstico.
Existe una enorme variación entre Unidades de Trasplante Renal en su consideración de las biopsias de protocolo, esto es, si deben ser usadas en la práctica clínica de ru-tina o seguir siendo un instrumento de investigación en centros seleccionados. La aproximación a este dilema depende de muchos factores que incluyen desde los regímenes de inmunosupresión que se utilizan, la participación en ensayos clínicos controlados de forma limitada o masiva, hasta los recursos disponibles. Ni que decir tiene que los considerandos sobre la seguridad y el partido que se saque a la prueba diagnóstica en los Departamentos de Anatomía Patológica son decisivos para la toma de decisión. Finalmente, se trataría de un análisis de costo-beneficio que relacionaría el número de eventos tratables diagnosticados y los injertos salvados en relación con el costo económico y de inconvenientes y complicaciones para el paciente.
Las biopsias con aguja para el seguimiento del trasplante renal, guiadas mediante ecografía con pistola automática y realizadas por personal experimentado, ofrecen un riesgo muy bajo de pérdida del injerto y un mínimo riesgo de morbilidad. Como se describe en este volumen de Biblioteca de Trasplantes Siglo XXI, la complicación más
frecuente y molesta es la hematuria macroscópica con obstrucción o no del uréter, siendo excepcionales las complicaciones más graves cuando se realiza la técnica se-gún las anteriores reglas expuestas. Parece generalmente aceptado que el riesgo es algo mayor cuando se practican biopsias por indicación clínica y cuando se utilizan agujas de un tamaño mayor del número 18.
Pero una de las mayores contraindicaciones para la biopsia de protocolo al igual que para la biopsia de indicación es no obtener una información clínica útil para tomar de-cisiones: la información histológica obtenida debe reflejar adecuadamente la situa-ción de actividad inmunológica o afectasitua-ción tóxica que de alguna manera proporcio-ne la base para el cambio terapéutico. Esta información se ve dificultada por la exis-tencia o no de nefropatólogo, o al menos un patólogo interesado y entrenado en la patología del trasplante renal, la variabilidad de la muestra y las diferencias entre ob-servadores.
No hay que olvidar que el rechazo celular leve-moderado es un proceso focal y que la interpretación de los patólogos puede diferir, sobre todo cuando se utiliza una escala de valoración como es la clasificación de Banff.
Por otro lado, el otro gran criterio a parte de la seguridad es la utilidad clínica de la biopsia. Lógicamente, este aspecto se valora en tanto en cuanto se detecte patología o, en una valoración más exigente, se detecte patología que sea modificable por una posible intervención terapéutica. La aparición del rechazo subclínico en el trasplante es absolutamente dependiente del tiempo desde la intervención: es máximo en el pri-mer mes y cae lentamente a lo largo del pripri-mer año. Existe una variación sustancial en la frecuencia que se ha publicado de rechazo subclínico que, probablemente, se re-laciona con esta variación en el momento de la realización de la biopsia, pero también en las características inmunológicas o étnicas de los receptores y de la inmunosupre-sión utilizada en cada Unidad.
Lo que resulta evidente actualmente es que la inmunosupresión es un condicionante a la hora de indicar biopsias de protocolo para la búsqueda diagnóstica de rechazo clí-nico, ya que la influencia del régimen inmunosupresor es decisiva, como se ha de-mostrado tanto en los trabajos clásicos del grupo australiano de Nankivell y Chapman como en las comunicaciones más recientes del Congreso Mundial de Boston de julio del 2006, realizadas por David Rush, pionero de las biopsias de proto-colo. Rush ha llegado a afirmar que la utilización de tacrolimus disminuye tanto la in-cidencia de esta complicación o de este diagnóstico que no estaría justificada la biopsia de protocolo para buscar esta afectación en pacientes que siguen el trata-miento expuesto. Por otra parte, la aparición de una patología inesperada puede ser importante y hay que considerar la posibilidad de la cada vez más emergente infec-ción por el virus BK.
8
Pero, independientemente de que el rechazo subclínico esté condicionado por el régimen utilizado, otro aspecto importante –y no del todo resuelto– es si una vez observada la infiltración celular en el injerto estable esto es significativo de daño actual o futuro, y si esta manifestación histológica y su posible modificación por la intervención terapeútica tiene incidencia o no en la supervivencia del paciente y del injerto a largo plazo. Existe muy poca experiencia en estudios controlados que analicen si el tratamiento o no del rechazo subclínico diagnosticado por biopsia de protocolo se sigue de una mejoría después de aplicar un tratamiento, y hay que re-ferirse casi siempre a las publicaciones iniciales de David Rush y Shisido, en las que la rama tratada con corticoides comparada con la rama no biopsiada y no trata-da tenía una mejor evolución.
Por lo tanto, la biopsia de protocolo es una clarísima situación de balance riesgo-be-neficio en la que hay que colocar en el platillo de la balanza del beriesgo-be-neficio la posibili-dad de mejorar el daño histológico, la supervivencia del injerto y del paciente, y los beneficios indirectos como reducir la inmunosupresión si se excluye el rechazo sub-clínico, la posible estratificación del riesgo inmunológico con la graduación de la in-munosupresión y el descubrimiento de diagnósticos no sospechados como glome-rulonefritis recurrente, nefrotoxicidad por anticalcineurínicos o infección viral del ti-po de la infección ti-por virus BK.
Como cabe esperar, este balance también depende del procedimiento en sí mismo y del riesgo de complicaciones, que son difíciles de comparar con el beneficio abso-luto obtenido del tratamiento del rechazo subclínico, porque no está bien cuantifica-do; las estimaciones se basan, en general, en el trabajo de Rush, en el que transcu-rridos cuatro años los pacientes a los que se les realizaron biopsias de protocolo presentaron 17 pérdidas de injerto menos por cada 100 pacientes. Si aplicamos aquí el NNT (número necesario de pacientes a tratar para obtener el beneficio) para prevenir una pérdida de injerto a los cuatro años, habría que incluir en el seguimien-to por biopsia de proseguimien-tocolo a seis pacientes siempre haciendo el cálculo de la preva-lencia de rechazo subclínico de la época de la ciclosporina, aproximadamente un 30%. No obstante, en la actualidad, el factor confundente para calcular este benefi-cio es la prevalencia descendente del rechazo agudo y del rechazo subclínico con la inmunosupresión actual que, con tacrolimus y MMF, se estima en un 2,6% de re-chazo subclínico y un 11% de rere-chazo borderline a los tres meses en receptores de raza blanca y de donante vivo.
Lo más generalmente aceptado es que la vigilancia mediante biopsias de protocolo es ética y clínicamente aceptable en Unidades de Trasplante en determinadas cir-cunstancias, como la función retrasada del injerto que precisa diálisis, pacientes hi-persensibilizados, trasplantes ABO incompatibles o cuando se está utilizando un end-point subrogado en los ensayos clínicos controlados.
Siguiendo de nuevo a Nankivell y Chapman en una publicación reciente, podemos decir que existen tres posibles estrategias que las Unidades de Trasplante tienen que considerar: no realizar biopsias de protocolo, efectuar biopsias sólo en pacientes de alto riesgo o seguir un protocolo universal en todos los pacientes de la serie. En cuanto a la primera, se trata de la posición que han adoptado hasta el momento la mayoría de las Unidades de Trasplante Renal del mundo, que asumen que el re-chazo subclínico es poco importante y puede ignorarse, o bien que es relevante pe-ro que se puede contpe-rolar mediante altas dosis de estepe-roides o aumentando la tera-péutica inmunosupresora. Los aspectos negativos de esta inmunosupresión inclui-rán un aumento de la nefropatía BK, infecciones nosocomiales y, probablemente, un incremento de los trastornos linferoproliferativos postrasplante. Sin embargo, evita los problemas de aceptación de esta técnica por parte del paciente cuando se encuentra asintomático, los costes económicos y de organización y, además, es acorde con las reglas para el seguimiento de los pacientes trasplantados editadas en el año 2000, que exponen que se requieren más datos antes de indicar de una forma generalizada la biopsia de protocolo.
La biopsia, en algunas situaciones, precisa realizar una selección de cuáles son los pacientes de alto riesgo y llevarla a cabo en algunos momentos determinados con un intervalo desde luego menor que las biopsias de protocolo seriadas generalizadas. Esto supone que es más económica y tiene claras desventajas como la selección de los pacientes y la definición de alto riesgo, que puede ser difícil o arbitraria. Esta se-lección se basa en que el rechazo subclínico puede ocurrir con mayor frecuencia y ser más severo que en los pacientes de riesgo normal o bajo; es muy clara cuando se eligen pacientes de extremo riesgo inmunológico; mientras que, en situaciones intermedias, esta predicción es mucho más imperfecta.
La decisión de aplicar una política de generalización y convertir la biopsia de proto-colo en un estándar de seguimiento –lógicamente, incluyendo excepciones o con-traindicaciones, como cuando hay un campo quirúrgico infectado o una obstrucción del sistema urinario, o bien el paciente está anticoagulado– tiene claras ventajas en la apreciación del rechazo subclínico no sospechado, ayuda a la toma de decisiones terapéuticas y descubre patologías encubiertas; sin embargo, presenta evidentes desventajas respecto del coste, el consumo de recursos y los inconvenientes para los pacientes, a pesar de que por cada procedimiento el coste y el riesgo no son ex-cesivos.
De cualquier forma, la decisión de aplicar de una forma más o menos generalizada las biopsias de protocolo en la rutina clínica exige unos criterios previos que tienen que seguirse estrictamente en la opinión de autores con tanta experiencia como los del grupo australiano.
10
PRERREQUISITOS PARA JUSTIFICAR LA BIOPSIA
DE PROTOCOLO DE RUTINA COMO HERRAMIENTA
DIAGNÓSTICA DEL RECHAZO SUBCLÍNICO
EN EL TRASPLANTE RENAL
(Nankivell BJ, Chapman JR. American Journal of Transplantation 2006)
1. Que la biopsia percutánea con aguja presente un bajo riesgo de pérdida del injerto y un ba-jo riesgo de morbilidad.
2. Que los resultados histológicos sean válidos y reflejen de forma adecuada la actividad in-munológica subclínica. Esto implica que:
a. Se tengan en cuenta la variabilidad biológica y los errores de muestra en las biopsias in-dividuales.
b. El tamaño de la muestra sea adecuado para el diagnóstico.
c. Los scores utilizados por los patólogos utilicen una herramienta estandarizada, como la clasificación de Banff.
d. Los resultados falsos negativos y positivos sean clínicamente aceptables.
3. Que la incidencia del rechazo subclínico sea suficientemente frecuente para justificar los esfuerzos diagnósticos.
4. Que el rechazo subclínico demuestre que es perjudicial para el injerto cuando se mira la función del injerto a largo plazo como daño histológico o por la disminución de la supervi-vencia del injerto.
5. Si se demuestra rechazo subclínico, que exista un tratamiento seguro y efectivo para re-vertirlo.
6. Que el control del rechazo subclínico conduzca a un beneficio neto para el receptor, que puede ser: directo pero diferido, como la mejoría del daño histológico, de la función del in-jerto o del paciente; o indirecto, como la posibilidad de reducir la inmunosupresión porque no hay rechazo subclínico o porque se descubren diagnósticos fortuitos potencialmente tratables como el virus BK o la nefrotoxicidad por anticalcineurínicos.
7. Que el riesgo-beneficio esté justificado en la mayoría de los receptores para ese grupo de-terminado.
8. Que exista un consenso clínico para la utilización de los recursos necesarios.
BIBLIOGRAFÍA RECOMENDADA
1. Nankivell BJ, Chapman JR. The significance of subclinical rejection and the value of protocol biopsies. American Journal of Transplantation 2006; 6: 2006-2012.
2. Rush D, Nickerson P, Gough J, McKenna R, Grimm P, Cheang M, Trpkov K, Solez K, Jeffery J. Beneficial effects of treatment of early subclinical rejection: a randomized study. J Am Soc Nephrol 1998; 9: 2129-34.
3. Rush D. No benefit of protocol biopsies in renal transplant patients receiving Tacrolimus and Mycophenolate Mofetil: a multicentre randomized study. Transplantation 2006; 82 (1): abstract 843.
4. Shishido S, Asanuma H, Nakai H, Mori Y, Satoh H, Kamimaki I, et al. The impact of re-peated subclinical acute rejection on the progression of chronic allograft nephropathy. J Am Soc Nephrol 2003; 14 (4): 1046-52.
5. Scholten EM, Rowshani AT, Cremers S, Bemelman FJ, Eikmans M, van Kan E, Mallat MJ, Florquin S, Surachno J, ten Berge IJ, Bajema IM, de Fijter JW. Untreated rejec-tion in 6-month protocol biopsies is not associated with fibrosis in serial biopsies or with loss of graft function. J Am Soc Nephrol 2006; 17: 2622-2632.
12
EARLY DIAGNOSIS OF CHRONIC
ALLOGRAFT NEPHROPATHY
BY MEANS OF PROTOCOL
BIOPSIES
J. Chapman
ROLE OF PROTOCOL BIOPSY EARLY
AFTER TRANSPLANTATION
The term “protocol biopsy” implies, in the context of renal transplantation, that biopsy is undertaken as a result of a pre-determined protocol and not because of a clinical event that has lead to the “need” for biopsy. The two processes have lead to very different answers over the years of development of renal transplantation. The clinically driven biopsy leads to a self-perpetuating result. Since biopsy is only performed at times of re-nal dysfunction, the results lead to the ability to distinguish between different causes of dysfunction –which essentially means distinguishing types of acute rejection from each other and from acute calcineurin inhibitor toxicity, or rare cases of pyelonephritis or vas-cular catastrophe. The clinically driven biopsy has not allowed distinction between the appearance of the kidney in times of acute dysfunction and times of stable function. It has been illuminating, and still remains surprising to some, that the histological data derived from protocol biopsies taken at times of stable function show a similar range of appearances to clinical biopsies. It has also required protocol biopsies to understand that the functional changes in renal allografts dramatically underestimate the severity of the pathological damage.
This chapter reviews the principles upon which the data are accrued from renal allograft biopsies specifically to understand the evolution of chronic allograft nephropathy (1, 2). It is possible to use these data to build and then test a model for understanding the
pa-thophysiology of chronic destruction of the renal transplant and the linkage between the underlying pathology and the observed functional changes.
THE RISK OF GRAFT COMPLICATIONS
In order to justify the use of protocol or surveillance percutaneous needle-core biopsies they must yield a low risk of complications including graft loss and morbidity such as ad-mission to hospital and hemorrhage. A variety of reports of the risk of major complica-tions including substantial bleeding, macroscopic hematuria with ureteric obstruction, pe-ritonitis or graft loss is approximately 1% (3-5). Minor complication include macroscopic hematuria in 3.5%, perirenal hematomas 2.5%, asymptomatic arterio-venous fistulas 7.3% and vasovagal reactions 0.5% (5).
Loss of the graft after protocol biopsy is in the region of 0.03%, based on various reports where the losses are recorded as: 0 from 2,127 biopsies (4), 1 from 1,171 (5), 0 of 328 (6), 0 from 1,037 (3), 0 from 961 biopsies (7), 0 from 277 (8), and 1 from 151 (9). The risk of renal allograft biopsy is increased when needles larger that 18G are used (6); when the biopsy is undertaken for clinical indications (presumably because the in-dication demands higher risks, or the graft is essentially unstable prior to the biopsy); and when the kidney is in an intraperitoneal position. Safety of protocol biopsies should thus be maximized through use of a skilled operator; mandatory ultrasound guidance immediately prior to biopsy to identify any unsuspected lesions such as urinary obs-truction or an arterio-venous fistula; and use of an automated spring loaded gun me-chanism rather than a manual cannula and trocar. Some operators suggest the use of a 16-gauge needle (10) and a single pass, rather than the 18-gauge needle favoured by many units but with more than one needle core sample (5). Certain exclusion cri-teria are also required to ensure that inappropriate risks are not taken. Table I details inclusion and exclusion criteria that should be considered prior to protocol biopsy. It would be appropriate for all of the inclusion criteria to be required to be met, but ex-clusion should require only a single criterion to be met. This is a conservative position designed to reduce the morbidity of the procedure undertaken with a lower yield of therapeutic decision making than occurs with acute clinically indicated biopsies. In par-ticular, the use of ultrasound not only localises the kidney accurately, but also identi-fies potential sources of complication, the commonest of which is partial ureteric obs-truction.
RELIABILITY OF PROTOCOL HISTOLOGY RESULTS
How good is the biopsy as a sample of the kidney? The reliability of a given result is re-lated to the amount of tissue obtained and to the variability if the histological finding with-in the kidney. Clearly very patchy events are seen with less reliability than homogeneous14
ones. Early and mild hyalinosis of the arterioles thus depends upon sampling sufficient arterioles and on hitting one with hyalinosis. Severe cellular ejection on the other hand is a diffuse process that can be seen throughout the kidney and can even be reliably im-plied on a section of medulla rather than cortex.
The best study of reproducibility of the Banff schema was undertaken by Furness et al. (11). The histologists eye is clearly quite inconsistent when assessing the area of in-volvement of a biopsy and may miss rare events such as occasional lymphocyte inva-sion of a tubule. The exact grading of tubulitis may thus be inaccurate and when missed may lead to change in the overall diagnosis of rejection status. Individual pathologists appear to be more consistent internally than when compared with each other, and in the Furness study remained rather resistant to change of their readings despite feedback on their results relative to the mean. Comparison of results between two centres found reasonable agreement for acute rejection diagnoses in another study (12) with a kappa statistic of 0.77 for agreement on the diagnosis of acute rejection. Veronese demons-trated similar levels of agreement for acute rejection (0.47 – 0.72) but borderline rejec-tion and fine grading of borderline rejecrejec-tion and individual Banff quantifiers was not so good (13).
EARLY DIAGNOSIS OF CHRONIC ALLOGRAFT NEPHROPATHY BY MEANS OF PROTOCOL BIOPSIES
Inclusion criteria
Deceased donor kidney allograft Informed consent
Approved clinical trial or standard clinical practice Ultrasound examination available
Skilled biopsy operator Exclusion criteria
Bleeding disorder
Use of anticoagulants including heparin, warfarin and aspirin Anatomical variation of graft position (e.g., overlying bowel) Anatomical variation of graft (e.g., arterio-venous malformation) Urinary obstruction
Requirement for sedation to undertake biopsy Previous complication from biopsy
To undertake protocol biopsy all of the inclusion criteria must be met and none of the exclusion criteria can be met.
A recent analysis of the reproducibility of the reading of arteriolar hyalinosis demonstra-tes the problem nicely (14). In this study the authors compared two protocols for reading –the standard Banff schema and a new schema developed by Mihatsch. The reproduci-bility of the new schema was better than the standard Banff schema (kappa statistic 0.67 versus 0.52 comparing 45 biopsies from 38 patients), but the information derived from the readings was less informative. In the new schema, reproducibility was improved through reduction of the frequency with which intermediate scoring grades were used. The number of times when scores of 1 and 2 were used dropped from 64 using Banff to 24 with the new system. This meant that biopsies were scored as 0 or 3 most of the time, which is little better than a binary decision of present/absent.
The essence of the problem is thus to find the balance between multiple grades of dis-tinction for a given event such as tubulitis or tubular atrophy, which extracts maximum amounts of information from the biopsy, and simple grading systems which are highly reproducible but uninformative. The compromise that is represented by the current Banff schema, does at least have widespread application across transplant programs and through many clinical trials. It is important to recognize that it remains limited by the degree of reproducibility at the margins and could suffer further refinement with benefit. The areas of contention remain, for example, the relevance of cellular infiltrate in areas of fibrosis. The current schema requires these to be ignored, but that leads to the potential for va-riability in interpretation of exactly what constitutes an area of fibrosis in which the infiltrate is to be ignored. Fortunately, the schema is under continuous revision and improvement, so it is reasonable to hold the view that, despite the areas of poor reproducibility, the Banff schema for the interpretation of renal allograft pathology is the best that we have available.
DEFINITIONS AND A MODEL OF CAN
Understanding the complexity of the entity of chronic allograft nephropathy (CAN) has taken many years, since unlike acute rejection, it cannot be easily identified clinically, is multifactorial in origin and evolves over years and not days or hours. The reliability and sensitivity of serum creatinine measurement contributed substantially to the ability to diagnose and treat acute rejection in the early years of transplantation and is still one of the most reliable tools that we use in the clinic to identify patients in need of further in-vestigation and change in management during the first few weeks after a renal trans-plant. Since the serum creatinine is also a fairly reliable indicator of chronic graft failure, as demonstrated by studies examining creatinine at one year as a predictor of graft loss, it has been natural to assume that monitoring the serum creatinine will identify grafts at risk from chronic graft failure and CAN. Experience has, however, taught us that this stra-tegy does not, on the whole, allow identification of CAN at a stage when intervention can substantially alter the course of events. It is central to the understanding of CAN to rea-lise why this is the case. It is also central to any strategies that might be effective in the
16
early identification and treatment of CAN. Our current model of CAN also demonstrates why treatment is not a very effective strategy and why prevention of CAN has to be the goal of all transplant units.
CAN is a diagnosis that has been arrived at through the pathologist’s microscope, ra-ther than the clinic. Papers from the 1970’s through to the mid 1990’s commonly used the term “chronic rejection” to describe the clinical phenomenon of slowly and in-exorably rising serum creatinine. Indeed this term can be found in papers from the past five years still loosely applied to chronic graft failure (15). It is important to un-derstand the difference between the starting points for each of these terminologies and the consequent confusions that apply when one term is used interchangeably with the other.
The term “CAN” arose from the Banff series of pathology conferences driven by such pathologists as Kim Solez, Robert Colvin, Michael Mihatsch, Daniel Serón and Lorraine Racusen (1). CAN is thus defined histologically, restricted to the renal allograft and is in-dependent of aetiology (16). The original intent of the definition was to bring some order into the chaotic descriptions that were available for a chronically damaged allograft. Unfortunately there remains some opportunity for confusion since the underlying aetio-logy may also be discernable in the biopsy specimen, such as calcineurin inhibitor (CNI) nephrotoxicity and through the addition to the diagnostic features of some facets with presumed immunological aetiology. Ample opportunity to confuse has been added by those who describe pre-transplant donor renal biopsies as having CAN –clearly an ab-surdity.
The features of CAN that are identified and scored in the Banff schema are tubular atrophy (Banff: ct) and interstitial fibrosis (Banff: ci). This should not be taken to mean that CAN is comprised of just these two appearances, nor that these are actually the most relevant to physiological, functional or prognostic indicators. It is simply that these two features are the most reliable, diffuse and easily visible changes in the biopsy, not subject to the vagaries of small samples that plague any definition relying on patchy changes in vessels or glomeruli. CAN grading from I to III simply identifies the propor-tion of the biopsy that is affected by these changes. It is unfortunate that the scoring of the chronic Banff “qualifiers” do not, in the current version, tie in well to the CAN grading. A revised version of the schema, with a name change to chronic sclerosing allograft nephropathy, may help us in the future. At the present time: CAN grade I requires ci1, ct1; CAN grade II requires moderate changes (ci2/ct2, or ci1/ct2, or ci2/ct1); while gra-de III requires severe changes (ci3/ct3, or ci2/ct3, or ci3/ct2). If there are none of the changes of “chronic rejection” such as vascular changes with disruption of the elasti-ca, inflammatory cells in the fibrotic intima and proliferation of myofibroblasts in the in-tima, the grade of CAN is qualified with an “a”, but if these changes are present then it is rates “b”.
EARLY DIAGNOSIS OF CHRONIC ALLOGRAFT NEPHROPATHY BY MEANS OF PROTOCOL BIOPSIES
The ground on which we have been able to build models of CAN is thus from histology and not from the clinical environment. Studies of biopsies taken in an attempt to diag-nose clinical events are also uselessly misleading since they are biased by the clinical overlay that comes with the decision to undertake the biopsy. It is thus only from proto-col biopsy studies that one can really build the conceptual models with which to test the-rapeutic interventions.
Reports of long term protocol renal allograft biopsy series were scarce until the past five years or so. The longest series was published from our group in Sydney (REF JASN 66-79) in which 120 recipients of simultaneous pancreas and kidney transplant recipients were biopsied at 0, 3 and 12 months and then annually thereafter to ten years. Our series, like that from the Mayo Clinic (17) which was predominantly from living donor transplants, star-ted with normal kidneys from young donors and very short cold ischemia times. We had previously published a shorter series of protocol biopsies taken from deceased donor grafts, which thus included the impact of preceding donor disease (9,18), while others have des-cribed the different impacts of varied immunosuppressive regimens (19-23).
Chronic allograft nephropathy is the final common pathway of different causes of renal allograft damage. That damage may have started in the hypertensive donor or through cold ischaemia and reperfusion injury, through acute rejection, acute or chronic CNI neph-rotoxicity and from sub-clinical rejection. It may be possible in a single biopsy to discern the relative influences of some of these factors, but in severe cases the biopsy is as uninformative as the “end stage kidney” that we are familiar with from native renal di-seases.
Our current working model is shown in Figure 1 and described below. We believe that it provides the foundation for understanding not only CAN, but also the relationships be-tween the histological appearances and the clinical perspectives (2). The new genomic techniques for elucidating changes at the molecular level may also potentially be incul-cated into explaining the model and the early evolution of CAN.
Subclinical rejection
Fibrosis of the kidney occurs in two separate phases (68), two thirds develops quite ra-pidly during the first year, with a slow and progressive accumulation beyond that time. Interestingly the interstitial fibrosis exceeds the tubular atrophy suggesting that it is rela-ted to more than simply the damage of tubules. The two strongest contenders for this ef-fect are ischaemia-reperfusion injury and acute allograft immune response –acute rejec-tion. In the Hannover protocol series of 258 patients (22) biopsied at 6, 12 and 16 weeks, CAN was present in 70 (37%) and absent in 120 at 26 weeks. The significant risk factors for CAN were receiving a deceased donor kidney, long cold ischaemia and acute rejec-tion. In the Westmead series acute tubular necrosis was predictive for CAN and the use
18
of mycophenolate mofetil compared with azathioprine reduced CAN (7). Sub-clinical re-jection was also a cause of interstitial fibrosis at twelve months (18) and CAN (25) in two independent series of
biopsies, the first in de-ceased donors of kidneys and the second in decea-sed donors of simulta-neous kidney pancreas transplants. The confir-mation that subclinical re-jection causes CAN, may lead to the conclusion that this phenomenon ex-plains the immune rela-ted risk factors for both CAN and long term graft survival. Sub-clinical re-jection is commonest in low intensity immuno-suppressive regimens and early after transplan-tation (Figure 2). It is
per-EARLY DIAGNOSIS OF CHRONIC ALLOGRAFT NEPHROPATHY BY MEANS OF PROTOCOL BIOPSIES
Transplant
Arteriosclerosis Arteriolar hyalinosis Tubular atrophy Interstitial fibrosis
Chronic donor disease Acute donor disease
Subclinical rejection/chronic humoral rejection CNI toxicity Rising creatinine Glomerular sclerosis
GRAFT LOSS
Acute rejection IschaemiaFigure 1.Model for the understanding the time dependent and inter-related features of chronic allograft nephropathy.
0 0.5 1 1.5
Mean Banff score
0 1 2 3 4 5 6-7
Years after transplantation
Interstitial inflammation Tubulitis
Figure 2.Prevalence of acute interstitial infiltration and tu-bulitis in protocol biopsies of renal allografts up to ten years post-transplantation.
sistent in sequential biopsies in only a small percentage, but in this group of patients it halves the measured GFR in only 18 months (25). In a formal randomised controlled trial of detection and treatment of early subclinical rejection, Rush and his colleagues de-monstrated a beneficial effect in terms of reduction of interstitial fibrosis and tubular atrophy at six months (26). More modern immunosuppressive regimens, including both tacrolimus and mycophenolate mofetil, have reduced the incidence of subclinical rejec-tion and also the power to demonstrate the efficacy of treating subclinical rejecrejec-tion (27). The Banff schema does not at present recognize inflammation in areas of fibrosis and thus assumes that this infiltrate has no predictive value since the damage has been done. There are now several series that demonstrate that this is not true and that sub-clinical inflammation in areas of fibrosis are indeed associated with the presence of increased fi-brosis in subsequent biopsies (28,29).
Taken together, these data clearly support the view that inflammatory cells in renal allo-grafts cause long term damage even in the absence of acute deterioration in graft function (30-36). In fact it is possible to support the contention that the damage is done because there is no associated rise in creatinine, allowing it to go undiagnosed and thus untreated.
CNI nephrotoxicity
The impact of CNI nephrotoxicity is the other major contender implicated in the deve-lopment of CAN. It is possible to recognize chronic CNI toxicity and CAN as separate his-tological entities and some have argued that this should always be the case (37). In the two year protocol biopsies from the US multicenter trial comparing cyclosporine and ta-crolimus (19), both the rate of development and the severity of CAN and chronic CNI to-xicity were indistinguishable between tacrolimus, and the two formulations of cyclospo-rine (38). In the Westmead series chronic CNI toxicity was identified in biopsies by the presence of at least two of: striped interstitial fibrosis; arteriolar hylainosis; and tubular micro-calcification. It was an almost universal finding by ten years after transplantation. Interstitial fibrosis and tubular atrophy are highly correlated and progress rapidly in the first year, thereafter progressing slowly but inexorably up to ten years and beyond.
Arteriolar hyalinosis
Arteriolar hyalinosis was present in 75% of patients by ten years. While implantation biop-sies may detect arteriolar hyalinosis in elderly, diabetic or hypertensive donors, in the se-ries of young donors of simultaneous kidney pancreas transplants that we reported, ar-teriolar hyalinosis first appeared, sometimes transiently in the first three to six months (38). Early changes were correlated with higher trough concentrations of cyclosporine and by preceding episodes of acute CNI toxicity. The longer term and permanent appear-ance of chronic CNI changes by five years post-transplant, was associated with higher
20
long term cyclosporine dosage with a cut off point at a maintenance dose of about 5 mg/ kg/day. The histological appearance that is classically described is of a nodular hyaline change in the wall of renal arterioles (14). In our data, however, this classical change of-ten gave way to more diffuse change with narrowing of the lumen and evidence of glo-merular ischaemia. The question thus arises as to whether the arteriolar change may be associated with hypertension or with diabetes in our series. We were able to dispel these two possibilities since treated hypertension succeeded the appearance of arteriolar hyali-nosis and did not preceed it as would be expected if it had a role in aetiology, also 60% of hypertensive and 68% of normotensive patients had arteriolar changes. With respect to the possibility of an effect from diabetes, the simultaneous glucose tolerance test per-formed with each protocol biopsy showed identical glucose handling in those with and those without arteriolar changes (38).
Glomerular disease
The final pathway for damage to the nephron is exhibited through glomerulosclerosis, with which irreversible loss of glomerular and nephron function occurs. Glomerular fil-tration rate is also closely linked to the glomerular sclerosis, especially as the number of functioning nephrons falls and glomerular hyperfiltration capacity is exceeded. There are at least three different mechanisms of destruction and pathologies that can be discerned in transplant glomeruli.
Recurrent glomerulonephritis
Occurs at different frequencies in different diseases and may account for a significant number of graft failures, such as in focal and segmental glomerular sclerosis (FSGS), or be essentially irrelevant such as in systemic lupus erythematosus (SLE) (15). No more will be said here about recurrent disease except to note that an increase in proteinuria by >500 mg/day or an absolute excretion rate of >1,500 mg/day are a reliable guide to the need for a diagnosis of the glomerular pathology (39), except possibly in patients treated with sirolimus or everolimus.
Chronic transplant glomerulopathy
In which the earliest signs, seen on electron microscopy include splitting of the base-ment membrane. This may be associated with the deposition of C4d and chronic anti-body mediated damage to the glomerulus (40). It has been well accepted since the 1960’s that antibody to HLA mediates hyperacute and forms of acute rejection. Transplant glo-merulopathy was recognised as early as 1963 with enlarged glomeruli, mesangial matrix expansion, changes in mesangial cells and splitting of both the glomerular and peritubu-lar basement membranes (41). Perhaps lulled into a false sense of security by the im-provement in crossmatching techniques, or perhaps by the confounding factors
sur-EARLY DIAGNOSIS OF CHRONIC ALLOGRAFT NEPHROPATHY BY MEANS OF PROTOCOL BIOPSIES
rounding the presence of anti-HLA antibody at the time of and subsequent to chronic allo-graft failure, it has taken many years to piece together the events that surround chronic humoral rejection. The turning point has come with the development of reliable techni-ques to detect the complement component C4d on the membrane of endothelial cells in the allograft and engagement with the Banff schema (42). Anti-HLA antibody is now known to predict a poor long term outcome (43) and the immune consequence of anti-body binding to complement on the endothelial cell surface to preceed transplant glo-merulopathy (44). Experimental evidence is now also linking these phenomena perhaps explaining one of the underlying mechanisms for vascular disease in the allograft (45). The recent demonstration of antibody to agrin –a component of the glomerular basement membrane– may explain the poor correlations between anti-HLA antibody and glomeru-lopathy (46). Transplant glomeruglomeru-lopathy is clearly identified as a cause for long term graft loss (47).
Ischaemic glomerular sclerosis
Associated with other features of chronic allograft nephropathy, seen progressively from three or four years post-transplantation (48). In the early post-transplant period glomeru-lar loss is associated with generalised ischaemic damage but is quite restricted in extent, at least in younger donors. The later and more significant phase of glomerular loss fol-lows as a consequence of earlier interstitial fibrosis and arteriolar hyalinosis (48). The se-verity of interstitial fibrosis at one year correlated with the sese-verity of glomerulosclerosis at four and five years, but even grafts with no interstitial fibrosis eventually developed glomerulosclerosis. A second factor influencing glomerular destruction was shown to be the severity of arteriolar hyalinosis, with substantial increases in the percentage of scle-rosed glomeruli for each grade increase in Banff designated arteriolar hyalinosis. These factors are familiar from prognostic studies in native renal diseases such as IgA nephro-pathy and it should come as little surprise to see the influence apparent in transplanted kidneys. Thus glomerulosclerosis is associated with both the results of acute and untrea-ted subclinical rejection, perhaps accounting for the correlations between chronic graft loss and early acute rejection noted in the 1980’s and 1990’s, as well as with indices of CNI nephrotoxicity, especially arteriolar hyalinosis.
Prevalence of chronic allograft nephropathy
The prevalence of CAN may only be determined from protocol biopsy of unselected pa-tients. All studies that report the prevalence of CAN based upon functional indices or se-lected histology driven by clinical indications dramatically underestimate the true preva-lence. There is no doubt that the prevalence of CAN depends upon many factors inclu-ding the donor age, indices of ischaemia and rejection, and upon the immunosuppres-sion used. One controlled study of cyclosporine compared to tacrolimus showed a pre-valence of CAN at two years to be 72.3% and 62.0% at two years respectively (19). The
22
prevalence was correlated with prior acute rejection episodes, acute nephrotoxicity and with donor age.
The Westmead series showed what was to some a remarkably high prevalence of CAN, with almost all grafts affected by CAN grade I by two years, and around 50% with CAN grade III by ten years (7) (Figure 3).
Steroid avoidance and use of either sirolimus or mycophenolate mofetil with tacrolimus lead to 20% grade II and III CAN at only 12 months in both African Americans and non-African Americans,
simi-lar to the Westmead data using predominantly cy-closporine therapy (49). Moreso et al. showed an even higher prevalence of CAN with 175 of 435 (40%) of grafts affected by six months (29). The precise estimate of CAN grade after at different ti-mes after transplantation varies with the study and to a certain extent also with the histologist, but the important and com-mon theme is that the le-vel of pathological impact is substantially underes-timated by the serum creatinine and other
func-tional measures in all studies. It is thus our contention that it is necessary to intervene early if one is to reduce the level of histological chronic allograft nephropathy.
Utility of diagnosing and detecting CAN early
One could legitimately ask the question: is it worth making the diagnosis of CAN? Is the-re a therapeutic utility to making the diagnosis? Is it possible to either the-reverse or pthe-revent deterioration in the histology once the diagnosis has been made? These questions are all predicated on therapeutic nihilism which dictates that once the damage has been done it cannot be undone. There is some justice in this approach, since sclerosed glomeruli will not spring back to life and atrophic tubules will not regenerate to normal morphology and function. The only conclusion that one can make is that therapy designed to halt the
EARLY DIAGNOSIS OF CHRONIC ALLOGRAFT NEPHROPATHY BY MEANS OF PROTOCOL BIOPSIES
Figure 3.Prevalence of chronic allograft nephropathy diag-nosed on protocol biopsy by the Banff criteria in 120 simul-taneous kidney pancreas transplant recipients.
progression of disease and therapy designed to remove the aetiological agents would have to be effective to justify the intervention.
The options available for prevention of graft damage are: treatment of subclinical rejec-tion; reversal of and prevention of further CNI nephrotoxicity, especially arteriolar hyali-nosis; prevention of ischaemia and selection of a younger donor. The latter strategies will not help after the transplant but there is evidence that treatment of subclinical rejection and switch from CNI based immunosuppression can make a difference.
Treatment of subclinical rejection
The diagnosis of subclinical rejection and the repeated and reliable demonstration of a correlation between subclinical cellular infiltrates and subsequent graft fibrosis, does not prove that it is possible to treat subclinical rejection with benefit. It was thus im-portant to see proof of this hypothesis in a single centre study from Canada (26). In this study Rush and his colleagues randomised half of the patients to protocol biopsy and treatment of subclinical rejection and the other half to treatment based upon clinical in-dices only. Interstitial fibrosis and tubular atrophy scores on biopsy at six months and serum creatinine at 24 months were statistically significantly superior in the protocol biopsy group, thus proving the hypothesis. In our own series, published recently, it was demonstrated that treatment of subclinical rejection diagnosed on one month protocol biopsy, eliminated differences in chronic fibrosis and tubular atrophy at three months, between those with and without subclinical rejection at one month (50). While this co-hort study again does not constitute formal proof of the utility of treating subclinical re-jection, it is encouraging that this therapy did seem to eliminate chronic damage which we have otherwise seen consistently for the past 15 or more years. A more recent for-mal trial by the Canadian group with a number of other North American centres has been reported in abstract form in 2006, but failed to demonstrate an impact because of the low incidence of subclinical rejection when using immunosuppression based upon ta-crolimus and mycophenloate mofetil (27). Interestingly there was a higher than expec-ted level of interstitial fibrosis in that study –perhaps exchanging subclinical rejection for CNI nephrotoxicity.
In conclusion, the weight of evidence favours the diagnosis and treatment of subclinical rejection, especially when using lower intensity immunosuppressive regimens, as a stra-tegy for reducing chronic allograft nephropathy.
Avoidance of CNI nephrotoxicity
One strategy for avoidance of nephrotoxicity is to avoid these agents entirely and a number of studies have attempted to do that by using combinations of sirolimus or eve-rolimus with mycophenolate mofetil and steroids with induction using a monoclonal
24
anti-Il2 receptor antibody (51). An alternative strategy has been studied since the early 1980’s, notably early conversion from a calcineurin inhibitor based regimen to one which eliminates the CNI after three, six or twelve months. It is not in the scope of this chap-ter to identify and review these studies which others have accomplished in formal meta-analyses (52, 53), but it is important to note that one of the more recent studies using sirolimus and corticosteroids as maintenance therapy was accompanied by protocol biopsies (54). In this study biopsies at implantation, 12 and 36 months were studied in a subset of patients that provided biopsy material at each time point. The most inter-esting feature of this study was that the group in which the CNI was eliminated had resolution of tubular atrophy between 12 and 36 months, without increased interstitial fibrosis. Thus the total CADI index of chronic damage was unchanged in the sirolimus treated patients, suggesting that the strategy might hold out some hopes for long term improvement in CAN, if effective and applicable clinical regimens can be developed to avoid the CNI’s.
Future opportunities
Clearly, very early diagnosis of events that may subsequently damage the graft might increase the effectiveness of interventions. The possibility that gene events will predict histology has been borne out in early studies from a number of groups including our own. Vitalone at the World Transplant Congress in Boston in July 2006 (55), presented data derived from Gene Microarrays demonstrating that genes associated with both CAN and rejection “turn on” –i.e., produce mRNA– earlier than the histological changes were apparent. If this is borne out in other laboratories and in other series of patients, it may offer a way of predicting the patients in whom intervention would be especially valuable.
SUMMARY
Chronic Allograft Nephropathy is the syndrome that has come to plague renal transplan-tation as the cause of most graft losses. It has been evident for as long as we have been transplanting the kidney, but in the early days it was overshadowed by acute rejection and mis-interpreted as an entirely immune related phenomenon termed “chronic rejec-tion”. It has been the source of most concern to clinical transplant programs for the past ten years, but the long term nature of the syndrome has meant that it has not been an easy target for research. Paradoxically, the pharmaceutical industry –which has the most to gain from long term use of particular agents– is most unlikely to fund the research trials that we will need to prove which are the best treatment options. Long term investments in this area are likely to be superseded before they have produced any answers. We are thus left with the individual transplant centre studies, implied answers from long term cohort studies using older pharmaceutical agents and new molecular genomic and pro-teomic technologies that have yet to deliver on their promise.
EARLY DIAGNOSIS OF CHRONIC ALLOGRAFT NEPHROPATHY BY MEANS OF PROTOCOL BIOPSIES
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EARLY DIAGNOSIS OF CHRONIC ALLOGRAFT NEPHROPATHY BY MEANS OF PROTOCOL BIOPSIES
DIAGNÓSTICO PRECOZ
DEL RECHAZO SUBCLÍNICO
MEDIANTE BIOPSIAS
DE PROTOCOLO
F. J. Moreso
DEFINICIÓN DE RECHAZO SUBCLÍNICO
El diagnóstico de rechazo subclínico se basa en la demostración histológica de hallazgos de rechazo agudo en una biopsia obtenida de un injerto renal con una función renal esta-ble. Las características histológicas, así como los tipos y grados de rechazo, se comen-tarán en el siguiente apartado. La definición de injerto renal con función estable no ha sido uniforme en distintos estudios llevados a cabo. Dado que desconocemos cuál es la me-jor función que puede alcanzar un injerto renal, se ha utilizado como criterio la diferencia entre la creatinina sérica en el momento de la biopsia y la creatinina sérica durante algún periodo próximo a la realización de la misma. En nuestra Unidad, se consideran injertos estables aquellos que muestran una diferencia entre la creatinina sérica el día de la biop-sia y la obtenida durante los 15-30 días antes y después de la biopbiop-sia inferior al 15% (1). De esta manera, se entiende que, en el caso de producirse un deterioro funcional supe-rior al 15% juntamente con hallazgos de rechazo agudo, se trataría de un caso de recha-zo clínico.
La primera descripción de hallazgos histológicos de rechazo agudo en injertos con fun-ción estable data de principios de la década de los 70 (2). A mediados de la década de los 80, se comunicó la presencia de signos de rechazo agudo en biopsias obtenidas en el momento del alta en pacientes trasplantados tratados con azatioprina o ciclosporina (3, 4). A pesar de estas aproximaciones iniciales, no ha sido hasta los últimos 15 años cuando la práctica de biopsias de protocolo en distintos centros ha permitido caracterizar la historia
natural de esta entidad, así como describir su epidemiología e implicaciones terapéuticas y pronósticas. Hay que tener en cuenta que las distintas reuniones del grupo de Banff han posibilitado desarrollar unos criterios estandarizados para la valoración de las biopsias (5-7), cuya aplicación por parte de los distintos centros ha hecho posible una descripción homogénea de las lesiones.
DIAGNÓSTICO DE RECHAZO SUBCLÍNICO
El diagnóstico de rechazo subclínico consiste en el estudio del material obtenido mediante punción-biopsia renal, el cual es procesado según las técnicas convencionales y valorado en función de los criterios aceptados internacionalmente.
Seguridad de la biopsia renal de protocolo
La biopsia del injerto renal se realiza habitualmente bajo control ecográfico utilizando agu-jas automáticas de los calibres 14 G, 16 G o 18 G, y se suelen obtener dos cilindros. Como toda técnica invasora, no está exenta de complicaciones y muchas Unidades de Trasplante prefieren no someter a ningún riesgo un injerto normofuncionante. Existen diversos es-tudios sobre la seguridad de la biopsia renal en el paciente trasplantado, pero han sido de gran utilidad estudios recientes que exploran la seguridad de la biopsia renal de protoco-lo (8, 9). De estos estudios se deduce que la pérdida del injerto renal es excepcional (una pérdida en más de 2.000 procedimientos) y que las complicaciones graves (que requie-ren transfusión y/o hospitalización) son inferiores al 1%.
Diagnóstico histológico
La biopsia se procesa en formalina, fija en parafina y se tiñe con las tinciones de hema-toxilina-eosina, ácido periódico de Schiff (PAS), tricrómico de Masson y plata metenami-na. La adecuación de la muestra obtenida, así como la valoración de las lesiones, se ba-san en los criterios definidos en la conferencia de consenso de Banff (5-7). Se aconseja teñir, al menos, siete láminas: tres con hematoxilina-eosina, tres con PAS y una con tri-crómico. Se consideran muestras adecuadas aquellas que contienen diez o más seccio-nes glomerulares y dos o más seccioseccio-nes arteriales. Son muestras marginales aquellas que contienen entre siete y nueve secciones glomerulares y, al menos, una sección ar-terial. Las muestras con menos de siete secciones glomerulares o sin secciones arteria-les no se consideran suficientes para una adecuada valoración.
Las lesiones histológicas se evalúan mediante una escala ordinal graduada de 0 a 3, en la cual 0 significa ausencia de lesión, 1 lesión leve, 2 lesión moderada y 3 lesión severa. Esta escala se utiliza para la valoración de las lesiones a nivel glomerular, intersticial, tubular y vascular, que se denominan glomerulitis, infiltrado intersticial, tubulitis y vasculitis, res-pectivamente, y que se abrevian por convenio como g, i, t y v, respectivamente.
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Según el esquema de Banff, el diagnóstico de cambios borderline se basa en la presen-cia de infiltrado interstipresen-cial leve (afecta al 10-25% de la corteza renal) y tubulitis leve (1-4 células inflamatorias por sección tubular). La existencia de infiltrado moderado (afecta en-tre el 25% y el 50% de la corteza renal) o severo (afecta a más del 50% del parénquima cortical) en asociación con tubulitis moderada (5-10 células inflamatorias por sección tu-bular) o severa (>10 células inflamatorias por sección tutu-bular) permite definir el rechazo agudo grados IA y IB, respectivamente. Finalmente, la presencia de arteritis intimal posi-bilita definir el rechazo agudo de grado II. En las Figuras 1-4 se muestran ejemplos de los distintos tipos de lesiones y grados de rechazo.
Diferencias entre rechazo agudo subclínico
y rechazo agudo clínico
Dado que los hallazgos histopatológicos en el rechazo agudo clínico y el rechazo agudo subclínico son indistinguibles, se han llevado a cabo distintos trabajos para detectar al-teraciones estructurales que justifiquen la presencia de lesiones de rechazo agudo sin deterioro funcional. Cabe destacar que en los injertos con función estable la presencia de rechazo vascular (grado II o III) es muy infrecuente, es decir, el rechazo vascular agu-do casi siempre se traduce en deterioro funcional. Para valorar la falta de deterioro
fun-DIAGNÓSTICO PRECOZ DEL RECHAZO SUBCLÍNICO MEDIANTE BIOPSIAS DE PROTOCOLO
Figura 1.Infiltrado inflamatorio difuso que afecta a más del 25% del área cortical (i2) con afectación predominantemente perivascular (hematoxili-na-eosina; x 100).
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BIOPSIA DE PROTOCOLO EN EL TRASPLANTE RENAL
Figura 3.Infiltrado linfomonocitario severo (i3) con presencia de eosinó-filos. Se observa tubulitis (t2) con destrucción de las membranas basales tubulares (hematoxilina-eosina; x 200).
Figura 2.Infiltrado inflamatorio leve con tubulitis leve (t1), entre una y cua-tro células inflamatorias por sección tubular (hematoxilina-eosina; x 400).
cional en los episodios de rechazo subclínico tubulointersticiales (cambios borderline y grado I), se ha estudiado la expresión fenotípica y el grado de activación de las células que infiltran el intersticio renal (10-13).
En un estudio que comparaba diez biopsias normales, 13 casos de rechazo subclínico y nueve de rechazo clínico, se evaluaron distintas subpoblaciones linfocitarias en el infil-trado intersticial (10). El número de células CD3 (linfocitos T) positivas era similar en los pacientes con rechazo clínico o subclínico. En cambio, se mostró una tendencia a un ma-yor número de células CD20 (linfocitos B), CD8 (linfocitos T citotóxicos) y CD68 (macró-fagos) positivas en aquellos pacientes con rechazo clínico que no alcanzaba significación estadística debido al pequeño tamaño de la muestra. En este mismo trabajo, se eviden-ció una mayor expresión de AIF-1 (factor inflamatorio del aloinjerto producido por los ma-crófagos activados), pero no se observó una mayor expresión de TNF-α (factor de ne-crosis tumoral), perforina o CD25 (receptor de interleucina 2). Por otra parte, este grupo de investigación ha demostrado que la expresión de RNA mensajero de perforina y otros mediadores de lesión es menor en los pacientes con rechazo subclínico que en los pa-cientes con rechazo clínico (11). Es decir, en el espectro entre la normalidad y el rechazo agudo con deterioro funcional se halla el rechazo subclínico, que muestra unas lesiones muy parecidas al rechazo clínico pero de menor intensidad.
DIAGNÓSTICO PRECOZ DEL RECHAZO SUBCLÍNICO MEDIANTE BIOPSIAS DE PROTOCOLO
Figura 4.Endotelialitis leve (v1) en las arterias de la zona intercorticome-dular (hematoxilina-eosina; x 100).
