University of Alberta, Edmonton, Canada a
University of Ċarhus, Denmark b
Baylor College of Medicine, Houston, USA c.
Key Words: Tubulitis, leukocyte common antigen, Leu-7
Corresponding Author: Luan D. Truong, Department of Pathology, The Methodist Hospital M.S. 205, Houston, Texas 77030, USA.
Beschorner et al  reported in 1985 that the presence of intratubular lymphocytes positive for the Leu-7 (CD57) antigen (i.e. Leu-7 tubulitis) was specific for rejection, and "quite helpful in the recognition of early acute renal allograft rejection". Most of the patients in this study did not receive cyclosporine treatment. Since that time, this monoclonal antibody has been used as a part of rejection diagnosis in several centers. However, the original observation has not been tested in a second full-length study, particularly in patients being treated with cyclosporine. Furthermore, others report no significant Leu-7+ renal infiltrates in acute rejection . The primary purpose of the present investigation was to see whether the specificity of Leu-7 tubulitis claimed by Beschorner et al can be confirmed, and to determine if the use of immunocytochemistry is justified in the routine assessment of renal transplant biopsies. We have also compared Leu-7 staining with that for leucocyte common antigen (i.e. LCA tubulitis). Tubulitis is known to occur in native kidney disease, therefore Leu-7 and LCA tubulitis were also assessed in cases of proliferative and nonproliferative glomerulonephritis (GN) and tubulointerstitial nephritis (TIN) in native kidney.
Glomerulitis (i.e. accumulation of MICs in glomeruli, with endothelial swelling) is assessed in the Banff schema . However, its significance in rejection and its relationship to other lesions is still undetermined. In the present study, accumulation of Leu-7+ and LCA+ cells in glomeruli was also assessed, and compared to similar accumulations in tubules and the interstitium.
Tissue Preparation and Staining Technique
The biopsies were fixed in B5 fixative (87 cases) or formalin (10 cases) for 2-4 hours, paraffin-embedded, and cut into four-micron consecutive sections for subsequent staining. In a preliminary study, it was noted that B5 fixative provided stronger immunocytochemical staining for the lymphoid cells than did formalin, but the quantitative aspects were similar for both. This observation has been previously noted for lymphoid tissue .
Aside from routine staining including hematoxylin and eosin, periodic acid-Schiff (PAS) and Masson's trichrome, tissue sections were also stained for LCA and Leu-7, using a standard avidin-biotin-peroxidase (ABC) technique with minor modifications . We used commercially available antibodies for LCA (mouse monoclonal antibody; clone PD7/26/16 and 2B11; Dako, Santa Barbara, Calif; dilution 1:30, one hour incubation) and Leu-7 (mouse monoclonal antibody; clone HNK-1; Becton-Dickinson, San Jose, Calif; prediluted, one hour incubation).
Methods of Evaluation
In each case, morphometric evaluation was done on three consecutive paraffin sections stained with PAS, LCA and Leu-7. The inflammatory cell infiltration within glomeruli, interstitium and tubules was separately assessed.
Glomeruli: The severity of glomerulitis was expressed as the glomerular inflammatory cell index (Gl Index). This was calculated by the ratio of the total number of MICs within the confines of Bowman capsules to the total number of available glomeruli. The PAS Gl, LCA Gl and Leu-7 Gl indices were similarly calculated from the appropriately-stained sections.
Interstitium: The degree of interstitial inflammation was reflected by the interstitial inflammatory index (In Index). Due to the variable number of cells identified with each stain, different methods of determining the In Index were used. For PAS, a subjective 0-4 PAS In Index was given to each case (0 = no inflammation, 1 = tubulointerstitial inflammation seen in 25% of the biopsy area, 2 = 26-50%, 3 = 51-75%, 4 = 76-100%). For LCA, the LCA In Index was similarly defined as the percentage area showing tubulointerstitial inflammation to the total area of the biopsy, but the values were accurately calculated by viewing the biopsy under the x10 objective of an American Optic Microscope, with a one cm2 reticule divided into 100 small squares attached to the eyepiece. The number of Leu-7+ cells was sufficiently small to enable counting of individual cells. Thus, the Leu-7 In Index was defined as the ratio of the total number of Leu-7+ cells identified outside the tubules to the total area of the biopsy, calculated by the reticule method described above.
Tubules: Quantitation of tubulitis was expressed as the tubulitis index (Tu Index). For both PAS and LCA, the Tu Index was calculated by counting, in areas with the most severe tubulointerstitial damage, the number of MICs which occupied the tubular profiles in eight microscopic fields (x40 objective, each 0.25mm 2 field delineated by the eyepiece reticule). The Leu-7 Tu Index was calculated as the ratio of the total number of Leu-7+ cells infiltrating the tubular epithelium to the total area of the biopsy. The entire biopsy was examined because Leu-7+ cells were in general quite scanty. MICs were rarely seen within the tubular lumina, but these cells were not used for the quantitation of tubulitis.
Statistical Method: Statistical correlation of the indices was performed using the Statview II program on a Macintosh SE/30 computer. The comparisons between groups were tested using analysis of variance followed by unpaired t-test between pairs of groups. Linear regression analysis and Spearman's rank test were used to test correlations between variables. Statistical significance was accepted at the 5% level. These correlations were performed only for the acute rejection group.
The morphometric evaluation is summarized in Table 2. The bottom row of this table shows analysis of variance (ANOVA) which compares the data for cases with acute rejection with the other diagnostic categories.
For all three staining methods (PAS, LCA and Leu-7), the Gl Index was significantly higher for the acute rejection category. Some glomerular mononuclear inflammatory infiltration was also present in proliferative GN (Fig.3), but was rare in other groups. The glomerulitis was most significantly elevated in rejection when it was detected by PAS (p<0.0001), followed by LCA (p<0.003), and least significantly elevated over the other categories when detected by Leu-7 (p<0.03). A wide variation was noted in the degree of glomerulitis from one glomerulus to the next in the same case, and from one case to the next in each of the diagnostic categories.
The degree of interstitial inflammation was most severe in the cases of rejection, followed by TIN; nevertheless, there was again a wide case-to-case variation in severity in each diagnostic category, especially for proliferative GN. The immunocytochemical In indices were more significantly elevated in acute rejection than the PAS In Index, with LCA and Leu-7 showing equal significance (p<0.0001). Interstitial Leu-7+ cells were seen in all diagnostic categories; in 100% of rejection cases, but also in 77% of cases with no acute rejection, 66% of proliferative GN, 68% of nonproliferative GN, and 69% of primary TIN. In each case, these cells accounted for a very small percentage of all the interstitial MICs.
Tubules For all three staining methods, tubulitis was significantly elevated in acute rejection (Figs.4-6) over other categories. The level of significance was maximal for PAS (p<0.0001), followed by LCA (p<0.001), and was least significant for Leu-7 (p<0.003). In severe acute rejection, infiltrating MICs were so numerous that tubular basement membranes were disrupted and some tubules were almost totally obliterated (Fig.5).
Table 3 details the percentages of cases with LCA tubulitis and Leu-7 tubulitis for each category. Cases from all categories displayed LCA tubulitis. It is interesting to note that while LCA tubulitis was found in 66% of transplant biopsies with no acute rejection, none of these cases showed Leu-7 tubulitis. In contrast, 100% of the acute rejection category had Leu-7 tubulitis, and the most severe Leu-7 tubulitis was seen in this category. Less severe Leu-7 tubulitis was also present in the native kidney diseases, i.e. 29% of proliferative GN, 18% of nonproliferative GN and 6% of primary TIN. It was noted that Leu-7 tubulitis was minimal in one case of TIN in which the interstitial inflammation was almost as severe as that in acute rejection.
The vasculitic lesions of acute vascular rejection contained LCA+ MICs. However, only occasional Leu-7+ cells were present in only one case of intimal arteritis. Leu-7+ cells were observed in the peritubular capillaries, and appeared much more frequent in this location in cases of rejection than in other diagnostic categories, although no quantitation of this phenomenon was performed.
Correlation of Parameters
Table 4 presents the statistical correlations in the acute rejection group of cases, and shows which of these are of significance. It can be seen that the LCA indices correlate with the PAS indices for all three features of glomerulitis, interstitial inflammation and tubulitis, although the significance was least for glomerulitis (p<0.02). Interstitial inflammation correlated with tubulitis when detected by LCA (p<0.01) and Leu-7 (p<0.002), but not when detected by PAS. Likewise, interstitial inflammation correlated with glomerulitis when detected by LCA (p<0.05) and Leu-7 (p<0.002), but not by PAS. Glomerulitis correlated with tubulitis when detected by PAS (p<0.005) and Leu-7 (p<0.02), but not by LCA. There was no correlation between LCA and Leu-7 for interstitial inflammation or for tubulitis, but LCA glomerulitis did correlate with Leu-7 glomerulitis (p<0.01).
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