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Leukocytes in the Graft Bed Associated with Corneal Graft Failure
Leukocytes in the Graft Bed Associated with Corneal Graft Failure Analysis by Immunohistology and Actuarial Graft Survival KERYN A. WILLIAMS, PhD, MICHELLE A. WHITE, BSc(Hons), JULIE K. ASH, BSc(Hons), DOUGLAS J. COSTER, FRACO Abstract: Immunohistochemical staining analysis using monoclonal antibodies was performed on 107 recipient corneas removed at graft. There were significantly more infiltrating cells bearing one or more of the leukocyte-common antigen, class II major histocompatibility complex antigens, various myeloidlineage markers, and a peripheral T cell marker, in the graft beds of those recipients who subsequently lost a corneal graft than there were in the graft beds of those for whom the outcome was successful. The hypothesis that large numbers of leukocytes in the recipient graft bed would be correlated with subsequent graft failure was examined by actuarial graft survival analysis. Recipients whose corneas contained fewer than 50 leukocyte-common antigen-positive cellsjmm 2 of corneal stroma showed a 3-year actuarial graft survival of 83%, compared with 39% in those whose corneas contained more than 50 such cellsjmm 2 . The corneal leukocyte count was a particularly useful prognostic indicator of outcome in those patients judged clinically to be at risk of graft failure. Ophthalmology 96:38-44, 1989
Well-established recipient risk factors for corneal graft failure include any combination of corneal neovascularization, l-J a history of anterior segment inflammation, 4 a history of raised intraocular pressure, 5·6 or failed previous corneal graft. 1•7 Irreversible graft rejection is a frequent, although by no means invariant, cause of graft failure in such recipients. Originally received: February 29, 1988. Revision accepted: September 13, 1988. From the Department of Ophthalmology, Flinders University of South Australia, Adelaide, Australia. Supported by the NH and MRC (grant 870684), the Ophthalmic Research Institute of Australia and the Wellcome Trust (Dr. Williams). Reprint requests to Keryn A. Williams, PhD, Department of Ophthalmology, Flinders Medical Centre, Bedford Park, S.A. 5042, Australia.
38
Almost 10 years ago, Tadros and colleagues8 observed that the fate of a corneal graft depended, to some extent, on the pathology of the graft bed into which the graft was placed. 8 We subsequently extended these findings to show that, in corneas of a small number of individuals with evidence of neovascularization or previous inflammatory episodes, the majority of cells present bore surface markers consistent with the phenotypes of various accessory cells (macrophages and dendritic cells), although the infiltrate was heterogeneous. 9 •10 Graft survival is best examined using actuarial analysis, a method which allows stratification of the patient pool and which accounts for varying lengths of follow-up. 11 • 12 We were interested to determine the predictive value of the host corneal leukocyte count on subsequent graft survival, using actuarial methods. A significant association was found between host leukocyte count at the time of graft, and graft outcome.
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PATIENTS AND METHODS PATIENTS
The Eye Clinic at the Flinders Medical Centre acts as a tertiary referral center for a city of approximately one million people. In consequence, a relatively high proportion of patients attending the clinic for assessment of corneal disease or opacity present with one or more of the established risk factors for the failure of a subsequent corneal graft. Of the 138 patients receiving a corneal graft between May 1983 and December 1986, 107 damaged recipient corneas excised at graft were available for study (77% ). All grafts were performed by the same surgeon and patients received consistent management. A rejection episode was diagnosed when a previously thin, clear graft became inflamed and subsequently developed edema with a loss of clarity. In many cases, either an endothelial or epithelial rejection line was observed. Rejection episodes were treated with topical 0.5% wt/vol prednisolone phosphate, applied hourly to the graft during the day for 14 days. If there was no response to topical therapy, then oral prednisolone ( 1 mg/kg/day) was prescribed for 7 days. Graft failure was defined as irreversible edema and opacity in a graft, whatever the reason for failure. SPECIMEN PREPARATION
Each excised cornea was cut into two pieces with a scalpel blade. One piece was fixed in buffered formalin and processed for routine histopathology, to allow confirmation of the clinical diagnosis. The other was fixed for 2 to 4 hours at 4 °C in paraformaldehyde-lysine-periodate. 13 It was washed sequentially in 7% wt/vol sucrose in phosphate-buffered saline (pH 7.4) followed by 15% wt/vol sucrose in the same buffer, for a total of 24 hours at 4 °C. Tissue was then immersed in water-soluble embedding medium (OCT 4583, Miles Scientific, IL) and snap-frozen in liquid nitrogen. Six micron-thick sections were cut in a cryostat at -20°C and transferred to chrome alum-subbed microscope slides. MOUSE ANTI-HUMAN MONOCLONAL ANTIBODIES
A panel of monoclonal antibodies was used. FMC 16 14 and W6/32 15 were used to mark monomorphic determinants of HLA class I antigens. FMC 4, 16 reactive with DP, DQ, and DR determinants, was used to mark HLA class II antigens. HLe-1 (Becton Dickinson, Mountain View, CA) and FMC 51 were used to mark leukocytecommon antigenic determinants; the former detects the 200 kdalton leukocyte-common antigen, 17 whereas the latter detects an antigen with a similar tissue distribution and the same molecular weight (Zola H, personal communication). T28 18 and Leu 4 (Becton Dickinson) 19 were used to mark the majority of human peripheral T cells. OKT 4 (Ortho Diagnostic Systems, Raritan, NJ) or Leu 3A (Becton Dickinson) were used to detect the CD4-pos-
itive peripheral T cell subset. 20·21 OKT 8 (Ortho Diagnostic Systems) was used to mark the CD8-positive T cell subsetY FMC 10 (detecting the CD 15 antigen), FMC 32 (detecting the CD 14 antigen), and FMC 34 were used to detect cells of the myeloid lineage. 23 •24 FMC 25 was used to detect the platelet antigen, gpiX/Ib. 25 Leuk 11 (Australian Monoclonal Development, Artarmon, New South Wales, Australia) was used to mark the transferrin receptor.26 HNK-1, produced from a cell-line obtained from the American Type Culture Collection (Rockville, MD), was used to detect some cells with natural killer activity. 26 Culture supernatant from the mouse myeloma cell line P3X63Ag8 (an IgGl/kappa-secreting line used to develop the nonsecreting fusion partner P3X63Ag8653, used to produce the FMC series of antibodies) was used as a negative control. IMMUNOPEROXIDASE STAINING
Immunoperoxidase staining was performed either by a four-layer method27 using DAKO products (DAKOPATTS, Copenhagen, Denmark) or with a Vectastain ABC kit designed to detect mouse IgG (Vector Laboratories, Burlingame, CA). 28 Any assay in which staining was seen with the negative control was discarded and the run repeated. FMC 16/ W6/32 were used as positive controls, because corneal epithelium carries HLA class I determinants. 29 Counts of positively stained cells in corneal stroma and epithelium were made as described previously;9 essentially, counts were converted to number of positive cells/mm2 of a 6-~m thick section after calculating the mean count for three randomly selected microscope fields. The observer was always unaware of the patients' indication for graft or of any of the clinical details. ACTUARIAL AND STATISTICAL ANALYSES
Actuarial graft survival curves were constructed according to Peto et al.ll· 12 Comparisons between curves were made using Gehan's generalized Wilcoxon chisquare statistics, a nonparametric alternative to the logrank test. There were three deaths in the patient cohort during the observation period; all patients died with functioning grafts, and trial times were adjusted accordingly. Two-by-two tables were examined using Fisher's exact test where the number of observations in any cell was less than five, and chi-square testing (with Yates' continuity correction) elsewhere. Student's t test was used to compare immunoperoxidase cell counts between patient groups.
RESULTS The primary indications for penetrating keratoplasty in the patients under study are presented in Table 1. Approximately one third of all grafts were performed for keratoconus, with failed previous graft, herpetic keratitis, corneal scarring, and aphakic bullous keratopathy accounting for the majority of the remainder. All grafts 39
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cleared and thinned satisfactorily in the immediate postoperative period and there were no primary graft failures. Immediately before graft, patients were divided into two groups on the basis of clinical assessment by a single surgeon. Those considered to be at a low risk for subsequent graft failure (37 patients) included patients with keratoconus, corneal dystrophy, and some of those suffering from corneal scarring. Patients in this group showed no evidence of corneal neovascularization, had no evidence of anterior segment inflammation or raised intraocular pressure, and had not previously rejected a corneal graft. The remaining 70 patients, all of whom had one or more risk factors for subsequent graft failure, were classified into a high-risk group. The assignment was made prospectively (before graft) in every instance. Immunoperoxidase cell counts performed on recipient corneas excised at graft are presented in Table 2, both for recipients with successful grafts, and for those with failed grafts at the time of analysis. The consulting ophthalmologist remained unaware of the results of the cell counts until the end of the study. Twenty-six grafts have thus far failed, with follow-up times ranging from 6 to 50 months. Mean cell counts in the group with failed grafts were significantly higher than in the group with successful grafts, for the antibodies FMC 51/HLe-1, FMC 4, FMC 34, FMC 32, FMC 10, and T28/Leu 4 (Student's t test, two-tailed). Cell counts for leukocyte-common antigenic determinants were used in subsequent analyses. Figure 1 shows a histogram of the number of counted leukocytes/mm2 of central cornea, for both the low and high clinical risk groups. Although the corneas from over 90% of patients
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Table 1. Indications for Graft No. of Patients
Primary Indication for Graft
(%)
34 (32) 29 (27) 11 (10)
Keratoconus Previous failed graft Corneal scarring, past history of HSV or HO Corneal scarring, no past history of HSV or HO Aphakic bullous keratopathy Pseudophakic bullous keratopathy Interstitial keratitis Fuchs' dystrophy Congenital corneal opacities Trauma, corneal perforation Fungal infection
7 (6)
11 (1 0) 2 (2) 6 (6) 2 (2) 2 (2) 2 (2) 1 (1)
107
Total HSV = herpes simplex virus keratitis; HO
= herpes zoster ophthalmicus.
in the low clinical risk group contained 50 or fewer leukocytes/mm2 cornea (a leukocyte being defined as a cell positive for HLe-1 or FMC 51), the spread for patients in the high clinical risk group was broad (median, 40 cells/ mm 2 ), with approximately 30% showing counts of more than 100 cells/mm2 (range, 2-1398 mm 2). A leukocyte of 50 cells/mm2 was chosen as an arbitrary cut-off point for low and high corneal cell counts. The numbers of successful and failed grafts for each clinical risk group, stratified according to leukocyte cell count at graft, are shown in Figure 2. In each group, a successful outcome tended to be associated with a low corneal count,
Table 2. lmmunoperoxidase-stained Recipient Corneas: Mean Cell Countsjmm 2 Central Cornea in the Groups with Surviving and Failed Grafts Successful Graft
Failed Graft
Antibody
Major Specificity
No. of Corneas Tested
FMC 51 I Hle-1 FMC 16/ W6/32 FMC 4
Leukocyte-common determinant MHC class I antigen MHC class II antigen
81 81 80
67 ± 176 49 ± 97 45 ± 67
26 26 26
155 ± 219t 159 ± 328 113 ± 110t
FMC 34 FMC 32 FMC 10
Granulocyte; monocyte Monocyte; C014 antigen Granulocyte; C015 antigen
59 66 20
19 ± 46 14 ± 33 7 ± 22
26 20 5
98 ± 199§ 51 ± 65t 46 ± 63t
T28/ Leu4 OKT 4/ Leu3A OKT 8 HNK-1
Peripheral T cell C04-positive T cell COB-positive T cell Natural killer cell
73 17 21 21
4 4 0.1 0
24 4 5 5
19 8 7 0.5
FMC 25 Leuk 11
Platelet gp IX/Ib antigen Transferrin receptor
28 21
0 ± 1 ±
SD = standard deviation; MHC = major histocompatibility complex. * In one instance, platelets were observed within a thrombus inside a capillary. t P< 0.05. t P< 0.001. § P< 0.01.
40
Cell Count (mean± SO)
No. of Corneas Tested
Cell Count (mean± SO)
± 11 ± 8 ± 0.4 ± 0 0 5
6* 5
± 27t ± 16 ± 12 ± 0.8
0 ± 0 12 ± 16
WILLIAMS et al
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HOST-TYPE CORNEAL LEUKOCYTES
CLINICAL LOW RISK GROUP LOW CUNICAL RISK
GRAFT OUTCOME
HIGH CLINICAL RISK
SUCCESS FAILURE
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CLINICAL HIGH RISK GROUP GRAFT OUTCOME 1-
z
SUCCESS
5
Figl. Histogram of the number ofleukocytes/mm 2 in the central stroma and epithelium of recipient cornea, excised at the time of graft.
FAILURE
(.)
....J ....J
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30
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and graft failure with a higher leukocyte count (P < 0.005 for the clinical low-risk group; P < 0.05 for the clinical high-risk group). Of the three patients considered to be at low risk for graft failure, but with corneal cell counts of higher than 50 cells/mm2 cornea it is noteworthy that two (suffering originally from keratoconus) have subsequently lost their grafts. The third, a neonate suffering from bilateral congenital corneal opacities, retains a clear graft after a 36month follow-up. Actuarial graft survival curves were then constructed for the subgroup of patients with corneal leukocyte counts of 0 to no more than 50 cells/mm2, and for the subgroup with greater than 50 cells/mm2 cornea, as shown in Figure 3. Three-year actuarial graft survival in the two groups was 83 and 39%, respectively (P < 0.0001 ). Patients in whom the indication for corneal transplantation is keratoconus generally enjoy excellent results after the grafe0 ; in this series, actuarial graft survival for the 34 patients with keratoconus was 95% at three years. Accordingly, actuarial survival curves for the two strata of low and high corneal cell counts were recalculated, with all patients suffering from keratoconus excluded from the analysis (Fig 4). The strata in Figure 4 correspond almost exactly to patients in the high clinical risk group, subdivided into those with low and high cell counts. Three-year actuarial graft survival was 74 and 41%, respectively (P < 0.001). Within the high clinical risk group, then, the number of leukocytes found to have infiltrated the recipient graft bed provides an excellent prognostic indicator of subsequent graft outcome. The causes of graft failure in the patient cohort are shown in Table 3. In ten instances, grafts failed from ir-
< w
z
a: 0
(.)
Fig 2. Two-by-two tables for the clinical low-risk and high-risk groups, showing corneal leukocyte cell count and graft outcome. The upper table was analyzed using Fisher's exact test, yielding P < 0.005. The lower table was analyzed using the chi-square test with Yates' continuity correction, yielding P < 0.05 (two-tailed).
reversible graft rejection. In four cases, irreversible rejection was considered to be the major cause of failure, with poorly controlled glaucoma being a contributory cause. In two other cases, irreversible rejection episodes occurred in eyes with intercurrent herpetic keratitis in the graft. Five grafts failed from glaucoma. One graft failed from the sequelae of repeated herpetic recurrences in the graft and another after total serous retinal detachment. There were two cases of suppurative keratitis in the graft (caused by Pseudomonas aeruginosa and Streptococcus sanguis respectively) and one case ofendophthalmitis from which mixed gram-negative organisms (Escherichia coli, Enter-
obacter cloacae, Hafnia a/veri, Pseudomonas acidovorans,
and Candida krusei) were isolated.
DISCUSSION When sections of damaged recipient cornea, excised at graft, were reacted with monoclonal antibodies and stain41
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~
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>50 CELLS/mm CORNEA n:32
:::d
> 50 CELLS/mm CORNEA n•35
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o~--_.----~----~----~----~
5
1
TRIAL TIME (YEARS POST-GRAFT) Fig 3. Actuarial corneal graft survival curves for patients with 50 leukocytes/mm2 or less of cornea, and for patients with greater than 50 cells/mm 2 cornea. n = number at risk in each stratum; P < 0.0001.
ing was visualized by an immunoperoxidase technique, counts of positively stained cells indicated that many specimens contained substantial leukocyte infiltrates, in confirmation of earlier studies. 8•9 Cell populations were heterogeneous, but in general, infiltrates were characterized by cells carrying a leukocyte-common antigen, together with class I MHC antigens. Many cells bore MHC class II antigens. Reactivity with antibodies detecting class I antigens was typically weak, probably reflecting partial destruction of the determinant by the fixative rather than low levels of expression, in that reactivity measured by immunofluorescence tests on unfixed human leukocyte was always strong. Many cells exhibited the characteristic morphology of the interstitial dendritic cell, 31 previously reported to be present in normal corneal epithelium32•33 and stroma9 ·34 ; such cells have been shown to move into the cornea after various traumatic stimuli. 9 •10•35 Substantial numbers of cells bearing various myeloid-lineage markers were also present, with macrophages usually outnumbering granulocytes. T cells were always a minor population: both CD4-positive and CD8-positive populations could be identified, but cells with a phenotypic marker of natural killer cells, reported to be able to kill corneal endothelial cells in vitro, 36 were infrequently present. The transferrin receptor, a marker of proliferating cells, including activated T cells, was seldom detected. There were virtually no platelets in any specimen. Most infiltrating cells, then, bore markers consistent with those of dendritic cells (i.e., CD45, class I and II MHC antigens, but none of the others examined) or macrophages (i.e., myeloid-lineage markers), and there were significantly more such cells in the graft beds of those recipients whose
42
NUMBER 1
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4
3
TRIAL TIME (YEARS POST -GRAFT) Fig 4. Actuarial corneal graft survival curves for the strata with low and high corneal cell counts, with patients suffering from keratoconus as the primary indication for graft excluded from both strata; n = number at risk in each stratum.
Table 3. Causes of Graft Failure in Patients with Low (s;50 cells/mm 2 cornea) and High (>50 cells/mm 2 cornea) Corneal Cell Counts No. of Failed Grafts Cause of Graft Failure Rejection Rejection and HSV recurrence Rejection and glaucoma Glaucoma HSV recurrence Suppurative keratitis Endophthalmitis Serous retinal detachment Total HSV
Low cell count 2 2
High cell count
8
2 2
2 3 1 2 1 1
8
18
= herpes simplex virus.
grafts eventually failed, than in those who retained clear grafts. Over 90% of corneas from patients in whom the presenting disease was keratoconus or one of the corneal dystrophies, contained relatively few leukocytes. Of interest was the observation that of three such patients with unusually high counts of infiltrating cells, two subsequently lost their graft. In the much larger subset of patients considered to be at higher risk of graft failure, a different distribution of corneal cell counts was observed, with approximately half of the corneal specimens examined containing quite substantial numbers. Actuarial graft survival
WILLIAMS et al
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HOST-TYPE CORNEAL LEUKOCYTES
analysis confirmed that low counts ofinfiltrating cells (::;;SO cells/mm2 central cornea) were associated with excellent graft survival (83% at 3 years), whereas the presence of more than 50 cells/mm2 cornea was associated with graft failure (39% actuarial survival at 3 years), irrespective of the clinical assessment before corneal transplantation. Of the 107 grafts followed, only three were performed in the face of active inflammation: the infiltrating cell count was high in each case, and all three grafts eventually failed. However, these cases are too few to account for the notably poor results obtained in the group with high corneal leukocyte counts, and there is argument as to whether corneal transplantation in the face of active inflammatory processes is associated with poorer graft survival than in cases where inflammation has been allowed to subside. 1•37 Although irreversible immunologic rejection, in some cases accompanied by glaucoma or recurrent herpetic disease, accounted for 61% of graft failures in this study, uncontrollable increases in intraocular pressure and the sequelae of infective episodes were also important reasons for graft loss. We have speculated that host-derived corneal dendritic cells and macrophages may present foreign alloantigen to host T cells, 9•10·38 especially where MHC determinants are shared between host and donor; some experimental evidence exists in support of this possibility.39.40 The presence of substantial numbers of accessory cells and, to a lesser extent T cells, in the bed of a corneal graft might then prejudice the survival of that graft. Clearly, however, rejection processes are irrelevant to graft failure in some instances. Glaucoma, in particular, is increasingly being recognized as an important cause of graft failure. 41 We suggest that in these cases, a high recipient corneal leukocyte count provides an accurate reflection of previous anterior segment disturbance or inflammation. A number of investigators have applied actuarial graft survival methodology to the identification of the prognostic factors that influence corneal graft survival. 1·4·7·42 ·43 Thus, for example, presenting disease, graft number, graft diameter, degree of HLA match, and degree of corneal vascularization at the time of graft are all important prognostic indicators. In the current study, virtually all of the 70 patients classified into the clinical high-risk group had evidence of corneal neovascularization at the time of graft; one third were receiving a second or subsequent graft. There were too few patients at risk to allow for sequential stratification according to all possible risk factors, but it is of interest that of the 47 patients in the clinical highrisk group receiving a first corneal graft, 2 of the 28 with low corneal cell counts have lost their grafts, compared with 9 of the 19 with high cell counts (P < 0.01, Fisher's exact test). Corneal cell count thus appears to be a useful prognostic indicator, independent of neovascularization and graft number. In conclusion, we have shown that the presence oflarge numbers of macrophages, dendritic cells, granulocytes, and (to a lesser degree) T cells in damaged recipient corneas before graft, is associated with subsequent corneal graft failure. The presence of more than 50 leukocytes/
mm 2 of central recipient cornea, at the time of the graft, provides an excellent prognostic indicator for graft loss.
ACKNOWLEDGMENTS The authors thank Dr. H. Zola for the gift of monoclonal antibodies.
REFERENCES 1. Vtilker-Dieben HJ. The Effect of Immunological and Non-Immunological Factors on Corneal Graft Survival. Dordrecht: Dr W Junk Publishers. 1984; 1-174. 2. Batchelor JR. Casey TA, Werb A. et al. HLA matching and corneal grafting. Lancet 1976; 1:551-4. 3. Khodadoust AA. The allograft rejection reaction: the leading cause of late failure of clinical corneal grafts. In: Corneal Graft Failure: Ciba Foundation Symposium 15. Amsterdam: Elsevier, 1973; 151-67. 4. Coster DJ. Factors affecting the outcome of corneal transplantation. Ann R Coli Surg Eng 1981; 63:91-7. 5. Goldberg DB, Schanzlin DJ, Brown Sl. Incidence of increased intraocular pressure after keratoplasty. Am J Ophthalmol1981; 92:372-7. 6. Polack FM. Graft rejection and glaucoma. Am J Ophthalmol 1986; 101:294-7. 7. Vtilker-Dieben HJ, D'Amaro J, Kok-Van Alphen CC. Hierarchy of prognostic factors for corneal allograft survival. Aust N Z J Ophthalmol 1987; 15:11-8. 8. Tadros ML, Hunter WS, Basu PK, et al. Prediction of graft fate from cytopathology of recipient button. Can J Ophthalmol1978; 13:102-4. 9. Williams KA, Ash JK, Coster DJ. Histocompatibility antigen and passenger cell content of normal and diseased human cornea. Transplantation 1985; 39:265-9. 10. Williams KA, Mann TS, Lewis M, Coster DJ. The role of resident accessory cells in corneal allograft rejection in the rabbit. Transplantation 1986; 42:667-71. 11. Peto R, Pike MC, Armitage P, et al. Design and analysis of randomized clinical trials requiring prolonged observation of each patient. I. Introduction and design. Br J Cancer 1976; 34:585-612. 12. Peto R, Pike MC, Armitage P, et al. Design and analysis of randomized clinical trials requiring prolonged observation of each patient. II. Analysis and examples. Br J Cancer 1977; 35:1-39. 13. Mclean IW, Nakane PK. Periodate-lysine-paraformaldehyde fixative. A new fixative for immunoelectron microscopy. J Histochem Cytochem 1974; 22:1077-83. 14. Zola H, McNamara PJ, Moore HA. et al. Maturation of human B lymphocytes-studies with a panel of monoclonal antibodies against membrane antigens. Clin Exp lmmunol1983; 52:655-64. 15. Barnstable CJ, Bodmer WF, Brown G, et al. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis. Cell1978; 14:9-20. 16. Beckman IGR, Bradley J, Brooks DA. et al. Human lymphocyte markers defined by antibodies derived from somatic cell hybrids. II. A hybridoma secreting antibody against an antigen expressed by human B and null lymphocytes. Clin Exp lmmunol 1980; 40:593-601. 17. Beverley PCL. Production and use of monoclonal antibodies in transplantation immunology. In: Toraine JL, Traeger J, Betuel H, et al, eds. Transplantation and Clinical Immunology. Vol. XI. Amsterdam: Excerpta Medica, 1986, 87-94. 18. Beverley PCL, Gallard RE. Distinctive functional characteristics of human "T" lymphocytes defined byE rosetting or a monoclonal anti-T cell antibody. Eur J lmmunol1981; 11:329-34.
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19. Ledbetter, JA, Evans RL, Lipinski M, et al. Evolutionary conservation of surtace molecules that distinguish T lymphocyte helper/inducer and cytotoxic/suppressor subpopulations in mouse and man. J Exp Med 1981; 153:310-23. 20. Reinherz EL, Kung PC, Goldstein G, Schlossman SF. Further characterization of human inducer T cell subset defined by monoclonal antibody. J lmmunol1979; 123:2894-6. 21. Evans RL, Wall OW, Platsoucas CD, et al. Thymus-dependent membrane antigens in man: inhibition of cell-mediated lympholysis by monoclonal antibodies to the TH2 antigen. Proc Natl Acad Sci USA 1981; 78:544-8. 22. Reinherz EL, Kung PC, Goldstein G, Schlossman SF. A monoclonal antibody reactive with the human cytotoxic/suppressor T cell subset previously defined by a heteroantiserum termed TH2 . J lmmunol1980; 124:1301-7.
35.
23. Zola H, McNamara P, Thomas M, et al. The preparation and properties of monoclonal antibodies against human granulocyte membrane antigens. Br J Haematol1981; 48:481-90.
36.
24. Hancock WW, Zola H, Atkins RC. Antigenic heterogeneity of human mononuclear phagocytes: immunohistologic analysis using monoclonal antibodies. Blood 1983; 62:1271-9. 25. Zola H, McNamara PJ, Beckman IGR, et al. Monoclonal antibodies against antigens of the human platelet surtace: preparation and properties. Pathology 1984; 16:73-8. 26. Abo T, Balch CM. A differentiation antigen of human NK and K cells identified by a monoclonal antibody. (HNK-1). J lmmunol1981; 127: 1024-9. 27. Hancock WW, Becker GJ, Atkins RC. A comparison of fixatives and immunohistochemical technicues for use with monoclonal antibodies to cell surtace antigens. Am J Clin Pathol 1982; 78:825-31. 28. Hsu S-M, Raine L, Fanger H. Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem 1981; 29:577-80. 29. Fujikawa LS, Colvin RB, Bhan AK, et al. Expression of HLA-A/B/C
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30. 31. 32.
33. 34.
37.
38. 39.
40. 41. 42.
43.
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and -DR locus antigens on epithelial, stromal, and endothelial cells of the human cornea. Cornea 1982; 1:213-22. Keates RH, Falkenstein S. Keratoplasty in keratoconus. Am J Ophthalmol1972; 74:442-4. Steinman RM, Nussenzweig MC. Dendritic cells: features and functions. lmmunol Rev 1980; 53:127-47. Rodrigues MM, Rowden G, Hackett J, Bakos I. Langerhans cells in the normal conjunctiva and peripheral cornea of selected species. Invest Ophthalmol Vis Sci 1981; 21:759-65. Gillette TE, Chandler JW, Greiner JV. Langerhans cells of the ocular surtace. Ophthalmology 1982; 89:700-11. Vantrappen L, Geboes K, Missotten L, et al. Lymphocytes and Langerhans cells in the normal human cornea. Invest Ophthalmol Vis Sci 1985; 26:220-5. Lang RM, Friedlaender MH, Schoenrock BJ. A new morphologic manifestation of Langerhans cells in guinea pig corneal transplants. Curr Eye Res 1981; 1:161-7. Opremcak EM, Whisler RL, Dangel ME. Natural killer cells against human corneal endothelium. Am J Ophthalmol1985; 99:524-9. Cabo LM, Coster OJ, Rice NSC, Jones BR. Prognosis and management of corneal transplantation for herpetic keratitis. Arch Ophthalmol 1980; 98:1755-9. Williams KA, Ash JK, Mann TS, et al. Cells infiltrating inflamed and vascularized corneas. Transplant Proc 1987; 19:2889-91. Lechler Rl, Batchelor JR. Restoration of immunogenicity to passenger cell-depleted kidney allografts by the addition of donor stain dendritic cells. J Exp Med 1982; 155:31-41. Sherwood RA, Brent L, Rayfield LS. Presentation of alloantigens by host cells. Eur J lmmunol1986; 16:569-74. Foulks GN. Glaucoma associated with penetrating keratoplasty. Ophthalmology 1987; 94:871-4. Bishop VLM, Robinson LP, Wechsler AW, Billson FA. Corneal graft survival: a retrospective Australian study. Aust N-Z J Ophthalmol1986; 14:133-8. Stulting RD, Sumers KD, Cavanagh HW, et al. Penetrating keratoplasty in children. Ophthalmology 1984; 91:1222-30.
Well-established recipient risk factors for corneal graft failure include any combination of corneal neovascularization, l-J a history of anterior segment inflammation, 4 a history of raised intraocular pressure, 5·6 or failed previous corneal graft. 1•7 Irreversible graft rejection is a frequent, although by no means invariant, cause of graft failure in such recipients. Originally received: February 29, 1988. Revision accepted: September 13, 1988. From the Department of Ophthalmology, Flinders University of South Australia, Adelaide, Australia. Supported by the NH and MRC (grant 870684), the Ophthalmic Research Institute of Australia and the Wellcome Trust (Dr. Williams). Reprint requests to Keryn A. Williams, PhD, Department of Ophthalmology, Flinders Medical Centre, Bedford Park, S.A. 5042, Australia.
38
Almost 10 years ago, Tadros and colleagues8 observed that the fate of a corneal graft depended, to some extent, on the pathology of the graft bed into which the graft was placed. 8 We subsequently extended these findings to show that, in corneas of a small number of individuals with evidence of neovascularization or previous inflammatory episodes, the majority of cells present bore surface markers consistent with the phenotypes of various accessory cells (macrophages and dendritic cells), although the infiltrate was heterogeneous. 9 •10 Graft survival is best examined using actuarial analysis, a method which allows stratification of the patient pool and which accounts for varying lengths of follow-up. 11 • 12 We were interested to determine the predictive value of the host corneal leukocyte count on subsequent graft survival, using actuarial methods. A significant association was found between host leukocyte count at the time of graft, and graft outcome.
WILLIAMS et al
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HOST-TYPE CORNEAL LEUKOCYTES
PATIENTS AND METHODS PATIENTS
The Eye Clinic at the Flinders Medical Centre acts as a tertiary referral center for a city of approximately one million people. In consequence, a relatively high proportion of patients attending the clinic for assessment of corneal disease or opacity present with one or more of the established risk factors for the failure of a subsequent corneal graft. Of the 138 patients receiving a corneal graft between May 1983 and December 1986, 107 damaged recipient corneas excised at graft were available for study (77% ). All grafts were performed by the same surgeon and patients received consistent management. A rejection episode was diagnosed when a previously thin, clear graft became inflamed and subsequently developed edema with a loss of clarity. In many cases, either an endothelial or epithelial rejection line was observed. Rejection episodes were treated with topical 0.5% wt/vol prednisolone phosphate, applied hourly to the graft during the day for 14 days. If there was no response to topical therapy, then oral prednisolone ( 1 mg/kg/day) was prescribed for 7 days. Graft failure was defined as irreversible edema and opacity in a graft, whatever the reason for failure. SPECIMEN PREPARATION
Each excised cornea was cut into two pieces with a scalpel blade. One piece was fixed in buffered formalin and processed for routine histopathology, to allow confirmation of the clinical diagnosis. The other was fixed for 2 to 4 hours at 4 °C in paraformaldehyde-lysine-periodate. 13 It was washed sequentially in 7% wt/vol sucrose in phosphate-buffered saline (pH 7.4) followed by 15% wt/vol sucrose in the same buffer, for a total of 24 hours at 4 °C. Tissue was then immersed in water-soluble embedding medium (OCT 4583, Miles Scientific, IL) and snap-frozen in liquid nitrogen. Six micron-thick sections were cut in a cryostat at -20°C and transferred to chrome alum-subbed microscope slides. MOUSE ANTI-HUMAN MONOCLONAL ANTIBODIES
A panel of monoclonal antibodies was used. FMC 16 14 and W6/32 15 were used to mark monomorphic determinants of HLA class I antigens. FMC 4, 16 reactive with DP, DQ, and DR determinants, was used to mark HLA class II antigens. HLe-1 (Becton Dickinson, Mountain View, CA) and FMC 51 were used to mark leukocytecommon antigenic determinants; the former detects the 200 kdalton leukocyte-common antigen, 17 whereas the latter detects an antigen with a similar tissue distribution and the same molecular weight (Zola H, personal communication). T28 18 and Leu 4 (Becton Dickinson) 19 were used to mark the majority of human peripheral T cells. OKT 4 (Ortho Diagnostic Systems, Raritan, NJ) or Leu 3A (Becton Dickinson) were used to detect the CD4-pos-
itive peripheral T cell subset. 20·21 OKT 8 (Ortho Diagnostic Systems) was used to mark the CD8-positive T cell subsetY FMC 10 (detecting the CD 15 antigen), FMC 32 (detecting the CD 14 antigen), and FMC 34 were used to detect cells of the myeloid lineage. 23 •24 FMC 25 was used to detect the platelet antigen, gpiX/Ib. 25 Leuk 11 (Australian Monoclonal Development, Artarmon, New South Wales, Australia) was used to mark the transferrin receptor.26 HNK-1, produced from a cell-line obtained from the American Type Culture Collection (Rockville, MD), was used to detect some cells with natural killer activity. 26 Culture supernatant from the mouse myeloma cell line P3X63Ag8 (an IgGl/kappa-secreting line used to develop the nonsecreting fusion partner P3X63Ag8653, used to produce the FMC series of antibodies) was used as a negative control. IMMUNOPEROXIDASE STAINING
Immunoperoxidase staining was performed either by a four-layer method27 using DAKO products (DAKOPATTS, Copenhagen, Denmark) or with a Vectastain ABC kit designed to detect mouse IgG (Vector Laboratories, Burlingame, CA). 28 Any assay in which staining was seen with the negative control was discarded and the run repeated. FMC 16/ W6/32 were used as positive controls, because corneal epithelium carries HLA class I determinants. 29 Counts of positively stained cells in corneal stroma and epithelium were made as described previously;9 essentially, counts were converted to number of positive cells/mm2 of a 6-~m thick section after calculating the mean count for three randomly selected microscope fields. The observer was always unaware of the patients' indication for graft or of any of the clinical details. ACTUARIAL AND STATISTICAL ANALYSES
Actuarial graft survival curves were constructed according to Peto et al.ll· 12 Comparisons between curves were made using Gehan's generalized Wilcoxon chisquare statistics, a nonparametric alternative to the logrank test. There were three deaths in the patient cohort during the observation period; all patients died with functioning grafts, and trial times were adjusted accordingly. Two-by-two tables were examined using Fisher's exact test where the number of observations in any cell was less than five, and chi-square testing (with Yates' continuity correction) elsewhere. Student's t test was used to compare immunoperoxidase cell counts between patient groups.
RESULTS The primary indications for penetrating keratoplasty in the patients under study are presented in Table 1. Approximately one third of all grafts were performed for keratoconus, with failed previous graft, herpetic keratitis, corneal scarring, and aphakic bullous keratopathy accounting for the majority of the remainder. All grafts 39
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cleared and thinned satisfactorily in the immediate postoperative period and there were no primary graft failures. Immediately before graft, patients were divided into two groups on the basis of clinical assessment by a single surgeon. Those considered to be at a low risk for subsequent graft failure (37 patients) included patients with keratoconus, corneal dystrophy, and some of those suffering from corneal scarring. Patients in this group showed no evidence of corneal neovascularization, had no evidence of anterior segment inflammation or raised intraocular pressure, and had not previously rejected a corneal graft. The remaining 70 patients, all of whom had one or more risk factors for subsequent graft failure, were classified into a high-risk group. The assignment was made prospectively (before graft) in every instance. Immunoperoxidase cell counts performed on recipient corneas excised at graft are presented in Table 2, both for recipients with successful grafts, and for those with failed grafts at the time of analysis. The consulting ophthalmologist remained unaware of the results of the cell counts until the end of the study. Twenty-six grafts have thus far failed, with follow-up times ranging from 6 to 50 months. Mean cell counts in the group with failed grafts were significantly higher than in the group with successful grafts, for the antibodies FMC 51/HLe-1, FMC 4, FMC 34, FMC 32, FMC 10, and T28/Leu 4 (Student's t test, two-tailed). Cell counts for leukocyte-common antigenic determinants were used in subsequent analyses. Figure 1 shows a histogram of the number of counted leukocytes/mm2 of central cornea, for both the low and high clinical risk groups. Although the corneas from over 90% of patients
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Table 1. Indications for Graft No. of Patients
Primary Indication for Graft
(%)
34 (32) 29 (27) 11 (10)
Keratoconus Previous failed graft Corneal scarring, past history of HSV or HO Corneal scarring, no past history of HSV or HO Aphakic bullous keratopathy Pseudophakic bullous keratopathy Interstitial keratitis Fuchs' dystrophy Congenital corneal opacities Trauma, corneal perforation Fungal infection
7 (6)
11 (1 0) 2 (2) 6 (6) 2 (2) 2 (2) 2 (2) 1 (1)
107
Total HSV = herpes simplex virus keratitis; HO
= herpes zoster ophthalmicus.
in the low clinical risk group contained 50 or fewer leukocytes/mm2 cornea (a leukocyte being defined as a cell positive for HLe-1 or FMC 51), the spread for patients in the high clinical risk group was broad (median, 40 cells/ mm 2 ), with approximately 30% showing counts of more than 100 cells/mm2 (range, 2-1398 mm 2). A leukocyte of 50 cells/mm2 was chosen as an arbitrary cut-off point for low and high corneal cell counts. The numbers of successful and failed grafts for each clinical risk group, stratified according to leukocyte cell count at graft, are shown in Figure 2. In each group, a successful outcome tended to be associated with a low corneal count,
Table 2. lmmunoperoxidase-stained Recipient Corneas: Mean Cell Countsjmm 2 Central Cornea in the Groups with Surviving and Failed Grafts Successful Graft
Failed Graft
Antibody
Major Specificity
No. of Corneas Tested
FMC 51 I Hle-1 FMC 16/ W6/32 FMC 4
Leukocyte-common determinant MHC class I antigen MHC class II antigen
81 81 80
67 ± 176 49 ± 97 45 ± 67
26 26 26
155 ± 219t 159 ± 328 113 ± 110t
FMC 34 FMC 32 FMC 10
Granulocyte; monocyte Monocyte; C014 antigen Granulocyte; C015 antigen
59 66 20
19 ± 46 14 ± 33 7 ± 22
26 20 5
98 ± 199§ 51 ± 65t 46 ± 63t
T28/ Leu4 OKT 4/ Leu3A OKT 8 HNK-1
Peripheral T cell C04-positive T cell COB-positive T cell Natural killer cell
73 17 21 21
4 4 0.1 0
24 4 5 5
19 8 7 0.5
FMC 25 Leuk 11
Platelet gp IX/Ib antigen Transferrin receptor
28 21
0 ± 1 ±
SD = standard deviation; MHC = major histocompatibility complex. * In one instance, platelets were observed within a thrombus inside a capillary. t P< 0.05. t P< 0.001. § P< 0.01.
40
Cell Count (mean± SO)
No. of Corneas Tested
Cell Count (mean± SO)
± 11 ± 8 ± 0.4 ± 0 0 5
6* 5
± 27t ± 16 ± 12 ± 0.8
0 ± 0 12 ± 16
WILLIAMS et al
30
D
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HOST-TYPE CORNEAL LEUKOCYTES
CLINICAL LOW RISK GROUP LOW CUNICAL RISK
GRAFT OUTCOME
HIGH CLINICAL RISK
SUCCESS FAILURE
1-
B
(.)
....J ....J
w (.) ....J
< w
z
:s: 50 /mrrf
34
0
34
>50 tmnf
1
2
3
35
2
37
a:
0
(.)
CLINICAL HIGH RISK GROUP GRAFT OUTCOME 1-
z
SUCCESS
5
Figl. Histogram of the number ofleukocytes/mm 2 in the central stroma and epithelium of recipient cornea, excised at the time of graft.
FAILURE
(.)
....J ....J
w
:S:50/mm'"
30
8
38
> 50/mm'
16
16
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and graft failure with a higher leukocyte count (P < 0.005 for the clinical low-risk group; P < 0.05 for the clinical high-risk group). Of the three patients considered to be at low risk for graft failure, but with corneal cell counts of higher than 50 cells/mm2 cornea it is noteworthy that two (suffering originally from keratoconus) have subsequently lost their grafts. The third, a neonate suffering from bilateral congenital corneal opacities, retains a clear graft after a 36month follow-up. Actuarial graft survival curves were then constructed for the subgroup of patients with corneal leukocyte counts of 0 to no more than 50 cells/mm2, and for the subgroup with greater than 50 cells/mm2 cornea, as shown in Figure 3. Three-year actuarial graft survival in the two groups was 83 and 39%, respectively (P < 0.0001 ). Patients in whom the indication for corneal transplantation is keratoconus generally enjoy excellent results after the grafe0 ; in this series, actuarial graft survival for the 34 patients with keratoconus was 95% at three years. Accordingly, actuarial survival curves for the two strata of low and high corneal cell counts were recalculated, with all patients suffering from keratoconus excluded from the analysis (Fig 4). The strata in Figure 4 correspond almost exactly to patients in the high clinical risk group, subdivided into those with low and high cell counts. Three-year actuarial graft survival was 74 and 41%, respectively (P < 0.001). Within the high clinical risk group, then, the number of leukocytes found to have infiltrated the recipient graft bed provides an excellent prognostic indicator of subsequent graft outcome. The causes of graft failure in the patient cohort are shown in Table 3. In ten instances, grafts failed from ir-
< w
z
a: 0
(.)
Fig 2. Two-by-two tables for the clinical low-risk and high-risk groups, showing corneal leukocyte cell count and graft outcome. The upper table was analyzed using Fisher's exact test, yielding P < 0.005. The lower table was analyzed using the chi-square test with Yates' continuity correction, yielding P < 0.05 (two-tailed).
reversible graft rejection. In four cases, irreversible rejection was considered to be the major cause of failure, with poorly controlled glaucoma being a contributory cause. In two other cases, irreversible rejection episodes occurred in eyes with intercurrent herpetic keratitis in the graft. Five grafts failed from glaucoma. One graft failed from the sequelae of repeated herpetic recurrences in the graft and another after total serous retinal detachment. There were two cases of suppurative keratitis in the graft (caused by Pseudomonas aeruginosa and Streptococcus sanguis respectively) and one case ofendophthalmitis from which mixed gram-negative organisms (Escherichia coli, Enter-
obacter cloacae, Hafnia a/veri, Pseudomonas acidovorans,
and Candida krusei) were isolated.
DISCUSSION When sections of damaged recipient cornea, excised at graft, were reacted with monoclonal antibodies and stain41
OPHTHALMOLOGY
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JANUARY 1989
~
2!:
> a:
:::>
(/)
t::
0.6
(.!'
u..
>
0
I:: :::d
0.4
~
:1.
~
~
0.6
<(
a:
~ ~
R:
m ~
0.2
~ 2
3
4
2
>50 CELLS/mm CORNEA n:32
:::d
> 50 CELLS/mm CORNEA n•35
0
0.2
o~--_.----~----~----~----~
5
1
TRIAL TIME (YEARS POST-GRAFT) Fig 3. Actuarial corneal graft survival curves for patients with 50 leukocytes/mm2 or less of cornea, and for patients with greater than 50 cells/mm 2 cornea. n = number at risk in each stratum; P < 0.0001.
ing was visualized by an immunoperoxidase technique, counts of positively stained cells indicated that many specimens contained substantial leukocyte infiltrates, in confirmation of earlier studies. 8•9 Cell populations were heterogeneous, but in general, infiltrates were characterized by cells carrying a leukocyte-common antigen, together with class I MHC antigens. Many cells bore MHC class II antigens. Reactivity with antibodies detecting class I antigens was typically weak, probably reflecting partial destruction of the determinant by the fixative rather than low levels of expression, in that reactivity measured by immunofluorescence tests on unfixed human leukocyte was always strong. Many cells exhibited the characteristic morphology of the interstitial dendritic cell, 31 previously reported to be present in normal corneal epithelium32•33 and stroma9 ·34 ; such cells have been shown to move into the cornea after various traumatic stimuli. 9 •10•35 Substantial numbers of cells bearing various myeloid-lineage markers were also present, with macrophages usually outnumbering granulocytes. T cells were always a minor population: both CD4-positive and CD8-positive populations could be identified, but cells with a phenotypic marker of natural killer cells, reported to be able to kill corneal endothelial cells in vitro, 36 were infrequently present. The transferrin receptor, a marker of proliferating cells, including activated T cells, was seldom detected. There were virtually no platelets in any specimen. Most infiltrating cells, then, bore markers consistent with those of dendritic cells (i.e., CD45, class I and II MHC antigens, but none of the others examined) or macrophages (i.e., myeloid-lineage markers), and there were significantly more such cells in the graft beds of those recipients whose
42
NUMBER 1
<(
>
<(
•
-I
;i
~ t
VOLUME 96
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5
4
3
TRIAL TIME (YEARS POST -GRAFT) Fig 4. Actuarial corneal graft survival curves for the strata with low and high corneal cell counts, with patients suffering from keratoconus as the primary indication for graft excluded from both strata; n = number at risk in each stratum.
Table 3. Causes of Graft Failure in Patients with Low (s;50 cells/mm 2 cornea) and High (>50 cells/mm 2 cornea) Corneal Cell Counts No. of Failed Grafts Cause of Graft Failure Rejection Rejection and HSV recurrence Rejection and glaucoma Glaucoma HSV recurrence Suppurative keratitis Endophthalmitis Serous retinal detachment Total HSV
Low cell count 2 2
High cell count
8
2 2
2 3 1 2 1 1
8
18
= herpes simplex virus.
grafts eventually failed, than in those who retained clear grafts. Over 90% of corneas from patients in whom the presenting disease was keratoconus or one of the corneal dystrophies, contained relatively few leukocytes. Of interest was the observation that of three such patients with unusually high counts of infiltrating cells, two subsequently lost their graft. In the much larger subset of patients considered to be at higher risk of graft failure, a different distribution of corneal cell counts was observed, with approximately half of the corneal specimens examined containing quite substantial numbers. Actuarial graft survival
WILLIAMS et al
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HOST-TYPE CORNEAL LEUKOCYTES
analysis confirmed that low counts ofinfiltrating cells (::;;SO cells/mm2 central cornea) were associated with excellent graft survival (83% at 3 years), whereas the presence of more than 50 cells/mm2 cornea was associated with graft failure (39% actuarial survival at 3 years), irrespective of the clinical assessment before corneal transplantation. Of the 107 grafts followed, only three were performed in the face of active inflammation: the infiltrating cell count was high in each case, and all three grafts eventually failed. However, these cases are too few to account for the notably poor results obtained in the group with high corneal leukocyte counts, and there is argument as to whether corneal transplantation in the face of active inflammatory processes is associated with poorer graft survival than in cases where inflammation has been allowed to subside. 1•37 Although irreversible immunologic rejection, in some cases accompanied by glaucoma or recurrent herpetic disease, accounted for 61% of graft failures in this study, uncontrollable increases in intraocular pressure and the sequelae of infective episodes were also important reasons for graft loss. We have speculated that host-derived corneal dendritic cells and macrophages may present foreign alloantigen to host T cells, 9•10·38 especially where MHC determinants are shared between host and donor; some experimental evidence exists in support of this possibility.39.40 The presence of substantial numbers of accessory cells and, to a lesser extent T cells, in the bed of a corneal graft might then prejudice the survival of that graft. Clearly, however, rejection processes are irrelevant to graft failure in some instances. Glaucoma, in particular, is increasingly being recognized as an important cause of graft failure. 41 We suggest that in these cases, a high recipient corneal leukocyte count provides an accurate reflection of previous anterior segment disturbance or inflammation. A number of investigators have applied actuarial graft survival methodology to the identification of the prognostic factors that influence corneal graft survival. 1·4·7·42 ·43 Thus, for example, presenting disease, graft number, graft diameter, degree of HLA match, and degree of corneal vascularization at the time of graft are all important prognostic indicators. In the current study, virtually all of the 70 patients classified into the clinical high-risk group had evidence of corneal neovascularization at the time of graft; one third were receiving a second or subsequent graft. There were too few patients at risk to allow for sequential stratification according to all possible risk factors, but it is of interest that of the 47 patients in the clinical highrisk group receiving a first corneal graft, 2 of the 28 with low corneal cell counts have lost their grafts, compared with 9 of the 19 with high cell counts (P < 0.01, Fisher's exact test). Corneal cell count thus appears to be a useful prognostic indicator, independent of neovascularization and graft number. In conclusion, we have shown that the presence oflarge numbers of macrophages, dendritic cells, granulocytes, and (to a lesser degree) T cells in damaged recipient corneas before graft, is associated with subsequent corneal graft failure. The presence of more than 50 leukocytes/
mm 2 of central recipient cornea, at the time of the graft, provides an excellent prognostic indicator for graft loss.
ACKNOWLEDGMENTS The authors thank Dr. H. Zola for the gift of monoclonal antibodies.
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35.
23. Zola H, McNamara P, Thomas M, et al. The preparation and properties of monoclonal antibodies against human granulocyte membrane antigens. Br J Haematol1981; 48:481-90.
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24. Hancock WW, Zola H, Atkins RC. Antigenic heterogeneity of human mononuclear phagocytes: immunohistologic analysis using monoclonal antibodies. Blood 1983; 62:1271-9. 25. Zola H, McNamara PJ, Beckman IGR, et al. Monoclonal antibodies against antigens of the human platelet surtace: preparation and properties. Pathology 1984; 16:73-8. 26. Abo T, Balch CM. A differentiation antigen of human NK and K cells identified by a monoclonal antibody. (HNK-1). J lmmunol1981; 127: 1024-9. 27. Hancock WW, Becker GJ, Atkins RC. A comparison of fixatives and immunohistochemical technicues for use with monoclonal antibodies to cell surtace antigens. Am J Clin Pathol 1982; 78:825-31. 28. Hsu S-M, Raine L, Fanger H. Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem 1981; 29:577-80. 29. Fujikawa LS, Colvin RB, Bhan AK, et al. Expression of HLA-A/B/C
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