Address Reprint Requests to: Professor Steen Olsen, M.D. Institute of Pathology Kommunehospitalet 8000 Ćarhus C, DenmarkKey Words: Renal allograft; Rejection; Proliferation rate; Ki-67 antigen
MIB-1 is an monoclonal antibody raised against recombinant fractions of the
Ki-67 gene [1, 2]. The antibody reacts with all phases of
the cell cycle except G0. It is effective in formaldehyde fixed paraffin wax
embedded material and has been found to be the best proliferation marker for
routine use in archival material .
We have used this method to study the proliferation rate in the interstitial infiltrate in renal graft biopsies from the first 90 days post transplant.
Standard immunosuppression was cyclosporine, azathioprine and prednisolone.
Rejection episodes were treated with methylprednisolone and monoclonal
antibodies (OKT 3).
Formalin fixed renal biopsies embedded in paraffin wax and cut into serial sections at 2 ”m were immunostained according to the method of Catoretti et al . Mouse monoclonal antibody MIB-1 against nuclear antigen Ki-67 was obtained from IMMUNOTECH, Marseille, France. The tissue sections were microwave treated with four cycles of 5 min each at 800 W. Lymph node was used as positive control for each batch of staining and a negative control (staining procedure without antibody) was added on each stained section.
Counting of cell nuclei of all as well as MIB-1 positive mononuclear leucocytes in the cortical interstitium was performed separately in dense, localized cell infiltrates and in tissue outside these infiltrates. The relative area of infiltrates was estimated by the point count method using total cortical area as reference. The counting was done with an unbiased counting frame  in a video image system using the GRID Stereological Package, Interactivision Inc., Silkeborg, Denmark. For statistical analyses the Wilcoxon signed rank method was used.
Mononuclear cells infiltrating in tubules (tubulitis) had MIB-1 positive nuclei in approximately the same relative number as cells in the infiltrates, but exact quantitation was not possible due to uncertainty of the precise location of some of these cells in relation to the tubular basement membrane in the immune staining.
Our study was performed to investigate if proliferation of infiltrating cells is related to histological rejection grade and to the fate of the graft. The appearance of a reliable method to mark proliferating cells in formalin fixed, paraffin embedded material made it possible to perform this investigation on a historical material.
The data from our quantitative study show that the number of interstitial cells per square mm in diffuse infiltrates is almost doubled in rejection compared with controls and that concentration of mononuclear cells is increased with a factor of ten in localized infiltrates. The percentage of cells in division is significantly increased from a level of 2.4 - 3 % in controls to more than 20 % in grade 2 and 3. We found no significant relation between these cell concentrations or proliferation rates with increasing Banff grades. This is interesting in view of the correlation which has been found between clinical and histological rejection [4, 8](4, 8).
Rejection treatment led to reversal of clinical rejection in all episodes except in 6 patients who lost their grafts within 1 year post transplant due to rejection. These patients had all had rejection episodes graded 2B or more. In the biopsies studied from patients who lost their grafts the number of infiltrating cells in diffuse as well as localized infiltrates and the relative number of proliferating cells were all within the same range as seen in biopsies from patients with functioning grafts after one year.
We conclude that counting of cell concentrations and proliferation indices do not add information to the diagnosis or grading of rejection which can not be achieved by the current histological Banff system. Decisive for prediction of poor graft prognosis is transition from grade 2A to 2B as previously shown by our group .
|Baseline||8||384 ±131||9.4 ±2.6||2.4||-||-||0|
|3 w plus fx||8||429 ±95||13.0 ±10.2||3.0||-||-||0|
|Acute Rejection Grade 1||8||706 ±251||96.9 ±90.9||13.7||7855 ±2947||723 ±807||9.2||12.6 ±7.6|
|Acute Rejection Grade 2A||6||649 ±423||222 ±173||23.3||6969 ±2534||950 ±1014||13.6||4.0 ±2.8|
|Acute Rejection Grade2B +3||10||691 ±255||143 ±75.9||20.7||7936 ±2169||987 ±1022||11.3||10.5 ±9.7|
|Rejected 1 year
(all grade 2B or 3)
|6||809 ±207||119 ±130||14.7||7962 ±2584||711 ±321||8.9||13.6 ±10.0|
Click here to go back.