LACK O F BLOOD GROUP ANTIGEN A ON HUMAN CORNEAL ENDOTHELIUM C. S T E P H E N F O S T E R , M.D.,
AND M A T H E A R. A L L A N S M I T H ,
Boston,
The homografted human cornea can elicit an immune response and can be destroyed by that response. Which antigenic determinants play a role in the stimulation of this immune response is unknown. Nelken and associates 1 report ed that preparations of whole, minced human corneas possess A and B blood group antigens. Herold 2 found these anti gens only in epithelium of 12 autopsied eyes; but questions arose as to whether his technique might have missed detec tion of the antigens, perhaps in low con centration, on the endothelium. Con flicting reports of the importance of ABO compatibility to keratoplasty outcome have been published. 3 "" 7 We describe herein our search for an easily detectable blood group antigen, A-antigen, on human corneal epithelium and endotheli um optimally prepared for preservation of the antigen. S U B J E C T S AND M E T H O D S
Tissue from 15 patients undergoing penetrating keratoplasty at the Massachu setts Eye and Ear Infirmary was used for this study. Ten of the patients had keratoconusc, three had corneal edema, and two had corneal scars from trauma. All pa tients were blood typed by the standard From the Department of Ophthalmology, Harvard Medical School; and the Department of Cornea Research, Eye Research Institute of Retina Founda tion, Boston, Massachusetts. This study was sup ported in part by Research Grants EY-00208 and EY-01552, Institutional National Research Service Award EY-07018, Biomedical Research Support Grant PHS5S07RR05527 from the National Eye Institute, National Institutes of Health; and in part by the Massachusetts Lions Eye Research Fund, Inc. Reprint requests to C. Stephen Foster, M.D., Eye Research Institute of Retina Foundation, 20 Stam ford St., Boston, MA 02114.
M.D.
Massachusetts
agglutination techniques. 8 Five of the pa tients were A positive, two were A nega tive, one was AB positive, and seven were O positive. Specimens of epithelium and of endo thelium from the 15 human corneal but tons removed at penetrating keratoplasty were placed on glass slides coated with silicone. The specimens were obtained and processed in the operating room im mediately after removal of the corneal tissue from the patient. Eight were stored at — 70'C and analyzed within ten days; seven were processed and examined im mediately. To insure adherence of cells to the slides, all tissue slides were briefly heatfixed immediately before specimen analy sis. Preliminary experiments with slides of type A human red blood cells showed that A-antigen was easily detectable with the fluorescent antibody technique and with the mixed agglutination technique after heat-fixation, even if the specimen has been stored at -70°C for three months before heat-fixation. The IgG fraction of human anti-A anti serum was prepared from normal human volunteers (blood type O positive) immu nized with purified human A substance. The antiserum was found, on screening with panels of test red blood cells, to contain no detectable antibodies to rhesus antigens or to antigens M, N, Lewis, Duffy, Kidd, and Kell. The antiserum was conjugated with fluorescein isothiocyanate. This antiserum was used both for the fluorescent antibody and for the mixed agglutination experiments. The protein content of the final preparation in 0.02M Tris-citrate buffer (pH 7.5) was 3.8 mg/ml and the fluorescein-protein ratio was 7.2 (xg of fluorescein per milligram of
AMERICAN JOURNAL O F OPHTHALMOLOGY 87:165-170, 1979
165
166
AMERICAN JOURNAL OF OPHTHALMOLOGY
protein. This ratio is higher than that used by most workers; but the antiserum did not stain non-type A red blood cells nonspecifically, and therefore was considered appropriate for the experiments. The highest dilution of this antiserum capable of promoting agglutination of type A human red blood cells was 1:8,192. The highest dilution capable of producing fluorescence of human type A red blood cells was 1:32. A control antiserum was prepared from the IgG fraction of human gamma globu lin from a blood type O positive donor. The protein content of this antiserum was 1.8 mg/ml and the fluorescein-protein ratio was 5.7 (xg of fluorescein per milli gram of protein. Specificity of the anti-A antiserum was confirmed by human type A red blood cell agglutination reactions, and by fluorescence microscopy of type A red blood cell smears stained with the antiserum. Red blood cells to be used as the test indicator cells in the mixed agglu tination experiments were obtained from blood type A, B, and O donors and pre pared in 0.5% phosphate-buffered saline suspensions. Corneal epithelial andendothelial spec imens from six of the patients were eval uated by the fluorescent antibody tech nique in masked fashion in duplicate. Undiluted antiserum was layered onto the specimen slides and allowed to remain at room temperature for 30 minutes. The slides were then washed twice in phos phate-buffered saline solution and were examined under a Zeiss fluorescence microscope fitted with a mercury bur ner, UG2 and BG12 excitation filters, and appropriate barrier filters. The degree of cell membrane fluorescence was graded on a scale of 0 to 4+ and was documented photographically. Tissue from the other nine patients was evaluated by the mixed agglutination technique. 9 Six slides of corneal epitheli al smears and six slides of corneal endo-
FEBRUARY, 1979
thelial smears were used for each of these nine patients. Anti-A antiserum was ap plied to three of the epithelial and three of the endothelial slides of each patient; control antiserum was applied to the re maining three slides of each tissue. After incubation at room temperature for one hour, the slides were washed four times with phosphate-buffered saline solution. Human indicator test red blood cell sus pensions (0.5% in phosphate-buffered sa line solution) were then layered onto the slides, which were incubated at room temperature for one hour. 10 Table 1 is an example of the tabular flow and data form used for the mixed agglutination studies of each specimen. In this example, epi thelial specimens from patient 12, a blood type A patient, exhibited a positive mixed agglutination reaction with type A red blood cells when anti-A antiserum was used, but not with other type red blood cells and not when control serum was used. At the end of the incubation period, the slides were allowed to drain for one hour to allow excess red blood cells to fall from the slides as recommended by Hogman. 11 The slides were covered with a cover slip secured with a drop of phosphate-buffered glycerol solution. Ex amination in masked fashion by phase contrast microscopy with a Leitz micro scope equipped with phase optics was performed. Adsorption of red blood cells TABLE 1 M I X E D AGGLUTINATION REACTION, HUMAN CORNEAL E P I T H E L I A L C E L L S AND HUMAN RED BLOOD C E L L S
Slide*
Antiserum
Indicator Cells
Reading
Epithelium Epithelium Epithelium Epithelium Epithelium Epithelium
Anti-A Anti-A Anti-A Control Control Control
A B O A B O
4+ ro.setting Negative Negative Negative Negative Negative
*Specimens from patient No. 12.
VOL. 87, NO. 2
LACK OF BLOOD GROUP ANTIGEN A
onto the corneal epithelial and endotheli al cells was evaluated. The criterion for rosetting was three or more red blood cells clustered against the epithelial or endothelial cell membrane. The results were documented photographically.
167
RESULTS
Fluorescent antibody technique—Aantigen could not be found on the endothelium of the corneas from blood type A or AB patients (Fig. 1). Corneal epithelial cell membranes from these patients
Fig. 1 (Foster and Allansmith). Top left, Corneal endothelial cells from blood type A patient. Fluorescei nated human anti-A antiserum used for staining. Note lack of positive fluorescence (background only, identical to picture obtained after staining with control antiserum). Top right, Corneal epithelial cells from blood type A patient. Fluoresceinated human anti-A antiserum used for staining. Note brilliant fluorescence of epithelial cell membrane. Bottom left, Corneal epithelial cells from blood type A patient. Fluoresceinated control antiserum used for staining. Note lack of fluorescence of epithelial cell membranes. Bottom right, Corneal endothelial cells from the blood type 0 patient. Fluoresceinated human anti-A antiserum used for staining. Note lack of fluorescence of endothelial cell membranes. Identical picture obtained when control antiserum was used for staining (fluorescence microscopy, x 1,000).
168
AMERICAN JOURNAL OF OPHTHALMOLOGY
showed brilliant fluorescence when the anti-A antiserum was used for staining. No fluorescence was seen when the con trol antiserum was used on the epithelium from these patients. Both endothelial and epithelial specimens from the type O pa tients did not show fluorescent staining with either antiserum (Table 2). Mixed agglutination technique—Blood group specific antigen A could not be found on the corneal endothelial cells of the four type A patients studied with this technique (Table 3) (Fig. 2). Antigen A was easily shown on the corneal epitheli al cells of these type A patients with this technique. When anti-A antiserum was applied to these epithelial cells, blood type A (but not type O or type B) indicator cells formed rosettes and multilayered red blood cell aggregates about the epithelial cells. This did not occur if control antiser um was used instead of anti-A antiserum. Both endothelial and epithelial speci mens from the four type O patients were negative. DISCUSSION
Using two separate techniques, differ ent from that used by Herold, 2 we have been unable to detect blood group anti gen A on the corneal endothelium of human corneas from blood type A donors. This is important because of the con flicting reports regarding the importance of ABO compatibility to corneal trans plant rejection in recipients with vasculTABLE 2 R E S U L T S O F F L U O R E S C E N T ANTIBODY STAINING O F HUMAN CORNEAL E P I T H E L I U M AND E N D O T H E L I U M FOR BLOOD GROUP A ANTIGEN* Blood
No. of
Group
Subjects
AntiA
Epithelium Control
AntiA
Endothelium Control
O A AB
2 3 1
0/2 3/3 1/1
0/2 0/3 0/1
0/2 0/3 0/1
0/2 0/3 0/1
* D a t a e x p r e s s e d as n u m b e r p o s i t i v e o v e r n u m b e r tested.
FEBRUARY, 1979 TABLE 3
R E S U L T S O F MIXED AGGLUTINATION REACTIONS O F HUMAN CORNEAL E P I T H E L I U M AND E N D O T H E L I U M AND B L O O D GROUP A R E D B L O O D C E L L S * Blood
No. of
Group
Subjects
AntiA
Epithelium Control
AntiA
Endothelium Control
O A
5 4
0/5 4/4
0/5 0/4
0/5 0/4
0/5 0/4
* D a t a e x p r e s s e d as n u m b e r p o s i t i v e o v e r n u m b e r tested.
arized corneas; because of the technical difficulties of detecting the fragile ABO antigens; and because the question of whether one of the major human transplan tation antigen systems, the ABO sys tem, is represented on corneal endotheli um is crucial to the understanding of which antigenic determinants on corneal endothelium stimulate an immune re sponse. Using the indirect immunofluorescence technique, Herold 2 showed blood group antigens A and B on human corneal epithelium, but could not detect these antigens on Bowman's membrane, stroma, Descemet's membrane, or endo thelium. We have now confirmed these findings by using direct immunofluorescence and the ultrasensitive mixed agglu tination reaction, two separate techniques, which are different from the technique used by Herold. Szulman 12 pointed out that A antigen deteriorates from a speci men within three to six hours if left in situ (as, for example, awaiting autopsy) and within 12 hours if stored at 4°C (as a surgical specimen, for example). One might argue that Herold failed to detect ABO antigens on corneal endothelium because he used eye bank eyes up to ten hours after death. Our tissue was process ed fresh or was frozen at — KfC, and the results were identical to those of Herold. We believe that the ABO blood group antigens can play no direct role in specific endothelial rejection, because they are not present on human corneal endothelium.
VOL. 87, NO. 2
LACK OF BLOOD GROUP ANTIGEN A
169
Fig. 2 (Foster and Allansmith). Top left, Corneal endothelial cells from blood type A patient. Slide has been treated with anti-A antiserum followed by washing and application of blood type A human red blood cells. Note lack of adsorption of red blood cells onto endothelial cell membranes. Top right, Corneal epithelial cells from blood type A patient. Slide has been treated with anti-A antiserum followed by washing and application of blood type A human red blood cells. Note dense pack and clustering of red blood cells in rosette formation around the epithelial cells. Bottom left, Corneal epithelial cells from blood type A patient. Slide has been treated with control antiserum followed by washing and application of blood type A human red blood cells. Note lack of adsorption of red blood cells onto epithelial cell membranes. Identical picture obtained if these epithelial cells were treated with anti-A antiserum followed by washing and application of blood type B or blood type 0 red blood cells. Bottom right, Corneal endothelial cells from blood type 0 patient. Slide has been treated with anti-A antiserum, followed by washing and application of blood type A human red blood cells. Note lack of adsorption of red blood cells onto endothelial cell membranes. Identical pictures obtained when blood type O and blood type B indicator red blood cells were used and when control antiserum was used (phase contrast microscopy, x500).
170
AMERICAN JOURNAL OF OPHTHALMOLOGY SUMMARY
Using direct immunofluorescence and mixed agglutination, we did not find blood group antigen A on human corneal endothelium from patients of blood type A or AB. The antigen was easily detected on the epithelium of these patients. We believe that the ABO blood group anti gens are probably absent on human cor neal endothelium, therefore they cannot play a direct role in specific corneal endothelial rejection. REFERENCES 1. Nelken, E., Nelken, D., Michaelson, I. C , and Gurevitch, J.: ABO antigens in the human cornea. Nature 177:840, 1956. 2. Herold, W.: Zum nachweis der blutgrappenantigene A und B in der menschlichen hornhaut mittels der immunofluoreszenztechnik. Klin. Monatsbl. Augenheilkd. 161:658, 1972. 3. Mailath, L., Stenszky, E., Albert, B., and Aszodi, L.: Compatibility examinations of donor's and recipient's blood group in connection with keratoplasty. Debrecen. Szemeszet. 109:112, 1972. 4. Bushmich, D. C , and Nikulina, N. B.: The
FEBRUARY, 1979
significance for the results of keratoplasty of select ing donor recipient pair according to group, type and rhesus characteristics. Oftalmol. Zh. 27:528, 1972. 5. Meyer, H. J.: The importance of blood factors in keratoplasty. Klin. Monatsbl. Augenheilkd. 158: 780, 1971. 6. Allansmith, M. R., Drell, D. W., Kajiyama, C , and Fine, M.: ABO blood groups and corneal trans plantation. Am. J. Ophthalmol. 79:493, 1975. 7. Batchelor, J. R., Casey, T. A., Gibbs, D. C , Lloyd, D. F., Werb, A., Prasad, S. S., and Jones, A.: HLA matching and corneal grafting. Lancet 1:551, 1976. 8. Landsteiner, K.: Uber agglutinationserscheinungen normalen menschlichen blutes. Klin. Wochenscher. 14:1132, 1901. 9. Coombs, R. R. A., Bedford, D., and Rouillard, L. M.: A and B blood group antigens on human epidermal cells demonstrated by mixed agglutina tion. Lancet 1:461, 1956. 10. Hogman, C. F.: Blood group antigens A and B determined by means of mixed agglutination on cultured cells of human fetal kidney, liver, spleen, lung, heart and skin. Vox. Sang. 4:319, 1959. 11. : The principle of mixed agglutination applied to tissue culture systems. Vox. Sang. 4:12, 1959. 12. Szulman, A. E.: The histological distribution of blood group substance A and B in man. J. Exp. Med. 111:785, 1960.
AND M A T H E A R. A L L A N S M I T H ,
Boston,
The homografted human cornea can elicit an immune response and can be destroyed by that response. Which antigenic determinants play a role in the stimulation of this immune response is unknown. Nelken and associates 1 report ed that preparations of whole, minced human corneas possess A and B blood group antigens. Herold 2 found these anti gens only in epithelium of 12 autopsied eyes; but questions arose as to whether his technique might have missed detec tion of the antigens, perhaps in low con centration, on the endothelium. Con flicting reports of the importance of ABO compatibility to keratoplasty outcome have been published. 3 "" 7 We describe herein our search for an easily detectable blood group antigen, A-antigen, on human corneal epithelium and endotheli um optimally prepared for preservation of the antigen. S U B J E C T S AND M E T H O D S
Tissue from 15 patients undergoing penetrating keratoplasty at the Massachu setts Eye and Ear Infirmary was used for this study. Ten of the patients had keratoconusc, three had corneal edema, and two had corneal scars from trauma. All pa tients were blood typed by the standard From the Department of Ophthalmology, Harvard Medical School; and the Department of Cornea Research, Eye Research Institute of Retina Founda tion, Boston, Massachusetts. This study was sup ported in part by Research Grants EY-00208 and EY-01552, Institutional National Research Service Award EY-07018, Biomedical Research Support Grant PHS5S07RR05527 from the National Eye Institute, National Institutes of Health; and in part by the Massachusetts Lions Eye Research Fund, Inc. Reprint requests to C. Stephen Foster, M.D., Eye Research Institute of Retina Foundation, 20 Stam ford St., Boston, MA 02114.
M.D.
Massachusetts
agglutination techniques. 8 Five of the pa tients were A positive, two were A nega tive, one was AB positive, and seven were O positive. Specimens of epithelium and of endo thelium from the 15 human corneal but tons removed at penetrating keratoplasty were placed on glass slides coated with silicone. The specimens were obtained and processed in the operating room im mediately after removal of the corneal tissue from the patient. Eight were stored at — 70'C and analyzed within ten days; seven were processed and examined im mediately. To insure adherence of cells to the slides, all tissue slides were briefly heatfixed immediately before specimen analy sis. Preliminary experiments with slides of type A human red blood cells showed that A-antigen was easily detectable with the fluorescent antibody technique and with the mixed agglutination technique after heat-fixation, even if the specimen has been stored at -70°C for three months before heat-fixation. The IgG fraction of human anti-A anti serum was prepared from normal human volunteers (blood type O positive) immu nized with purified human A substance. The antiserum was found, on screening with panels of test red blood cells, to contain no detectable antibodies to rhesus antigens or to antigens M, N, Lewis, Duffy, Kidd, and Kell. The antiserum was conjugated with fluorescein isothiocyanate. This antiserum was used both for the fluorescent antibody and for the mixed agglutination experiments. The protein content of the final preparation in 0.02M Tris-citrate buffer (pH 7.5) was 3.8 mg/ml and the fluorescein-protein ratio was 7.2 (xg of fluorescein per milligram of
AMERICAN JOURNAL O F OPHTHALMOLOGY 87:165-170, 1979
165
166
AMERICAN JOURNAL OF OPHTHALMOLOGY
protein. This ratio is higher than that used by most workers; but the antiserum did not stain non-type A red blood cells nonspecifically, and therefore was considered appropriate for the experiments. The highest dilution of this antiserum capable of promoting agglutination of type A human red blood cells was 1:8,192. The highest dilution capable of producing fluorescence of human type A red blood cells was 1:32. A control antiserum was prepared from the IgG fraction of human gamma globu lin from a blood type O positive donor. The protein content of this antiserum was 1.8 mg/ml and the fluorescein-protein ratio was 5.7 (xg of fluorescein per milli gram of protein. Specificity of the anti-A antiserum was confirmed by human type A red blood cell agglutination reactions, and by fluorescence microscopy of type A red blood cell smears stained with the antiserum. Red blood cells to be used as the test indicator cells in the mixed agglu tination experiments were obtained from blood type A, B, and O donors and pre pared in 0.5% phosphate-buffered saline suspensions. Corneal epithelial andendothelial spec imens from six of the patients were eval uated by the fluorescent antibody tech nique in masked fashion in duplicate. Undiluted antiserum was layered onto the specimen slides and allowed to remain at room temperature for 30 minutes. The slides were then washed twice in phos phate-buffered saline solution and were examined under a Zeiss fluorescence microscope fitted with a mercury bur ner, UG2 and BG12 excitation filters, and appropriate barrier filters. The degree of cell membrane fluorescence was graded on a scale of 0 to 4+ and was documented photographically. Tissue from the other nine patients was evaluated by the mixed agglutination technique. 9 Six slides of corneal epitheli al smears and six slides of corneal endo-
FEBRUARY, 1979
thelial smears were used for each of these nine patients. Anti-A antiserum was ap plied to three of the epithelial and three of the endothelial slides of each patient; control antiserum was applied to the re maining three slides of each tissue. After incubation at room temperature for one hour, the slides were washed four times with phosphate-buffered saline solution. Human indicator test red blood cell sus pensions (0.5% in phosphate-buffered sa line solution) were then layered onto the slides, which were incubated at room temperature for one hour. 10 Table 1 is an example of the tabular flow and data form used for the mixed agglutination studies of each specimen. In this example, epi thelial specimens from patient 12, a blood type A patient, exhibited a positive mixed agglutination reaction with type A red blood cells when anti-A antiserum was used, but not with other type red blood cells and not when control serum was used. At the end of the incubation period, the slides were allowed to drain for one hour to allow excess red blood cells to fall from the slides as recommended by Hogman. 11 The slides were covered with a cover slip secured with a drop of phosphate-buffered glycerol solution. Ex amination in masked fashion by phase contrast microscopy with a Leitz micro scope equipped with phase optics was performed. Adsorption of red blood cells TABLE 1 M I X E D AGGLUTINATION REACTION, HUMAN CORNEAL E P I T H E L I A L C E L L S AND HUMAN RED BLOOD C E L L S
Slide*
Antiserum
Indicator Cells
Reading
Epithelium Epithelium Epithelium Epithelium Epithelium Epithelium
Anti-A Anti-A Anti-A Control Control Control
A B O A B O
4+ ro.setting Negative Negative Negative Negative Negative
*Specimens from patient No. 12.
VOL. 87, NO. 2
LACK OF BLOOD GROUP ANTIGEN A
onto the corneal epithelial and endotheli al cells was evaluated. The criterion for rosetting was three or more red blood cells clustered against the epithelial or endothelial cell membrane. The results were documented photographically.
167
RESULTS
Fluorescent antibody technique—Aantigen could not be found on the endothelium of the corneas from blood type A or AB patients (Fig. 1). Corneal epithelial cell membranes from these patients
Fig. 1 (Foster and Allansmith). Top left, Corneal endothelial cells from blood type A patient. Fluorescei nated human anti-A antiserum used for staining. Note lack of positive fluorescence (background only, identical to picture obtained after staining with control antiserum). Top right, Corneal epithelial cells from blood type A patient. Fluoresceinated human anti-A antiserum used for staining. Note brilliant fluorescence of epithelial cell membrane. Bottom left, Corneal epithelial cells from blood type A patient. Fluoresceinated control antiserum used for staining. Note lack of fluorescence of epithelial cell membranes. Bottom right, Corneal endothelial cells from the blood type 0 patient. Fluoresceinated human anti-A antiserum used for staining. Note lack of fluorescence of endothelial cell membranes. Identical picture obtained when control antiserum was used for staining (fluorescence microscopy, x 1,000).
168
AMERICAN JOURNAL OF OPHTHALMOLOGY
showed brilliant fluorescence when the anti-A antiserum was used for staining. No fluorescence was seen when the con trol antiserum was used on the epithelium from these patients. Both endothelial and epithelial specimens from the type O pa tients did not show fluorescent staining with either antiserum (Table 2). Mixed agglutination technique—Blood group specific antigen A could not be found on the corneal endothelial cells of the four type A patients studied with this technique (Table 3) (Fig. 2). Antigen A was easily shown on the corneal epitheli al cells of these type A patients with this technique. When anti-A antiserum was applied to these epithelial cells, blood type A (but not type O or type B) indicator cells formed rosettes and multilayered red blood cell aggregates about the epithelial cells. This did not occur if control antiser um was used instead of anti-A antiserum. Both endothelial and epithelial speci mens from the four type O patients were negative. DISCUSSION
Using two separate techniques, differ ent from that used by Herold, 2 we have been unable to detect blood group anti gen A on the corneal endothelium of human corneas from blood type A donors. This is important because of the con flicting reports regarding the importance of ABO compatibility to corneal trans plant rejection in recipients with vasculTABLE 2 R E S U L T S O F F L U O R E S C E N T ANTIBODY STAINING O F HUMAN CORNEAL E P I T H E L I U M AND E N D O T H E L I U M FOR BLOOD GROUP A ANTIGEN* Blood
No. of
Group
Subjects
AntiA
Epithelium Control
AntiA
Endothelium Control
O A AB
2 3 1
0/2 3/3 1/1
0/2 0/3 0/1
0/2 0/3 0/1
0/2 0/3 0/1
* D a t a e x p r e s s e d as n u m b e r p o s i t i v e o v e r n u m b e r tested.
FEBRUARY, 1979 TABLE 3
R E S U L T S O F MIXED AGGLUTINATION REACTIONS O F HUMAN CORNEAL E P I T H E L I U M AND E N D O T H E L I U M AND B L O O D GROUP A R E D B L O O D C E L L S * Blood
No. of
Group
Subjects
AntiA
Epithelium Control
AntiA
Endothelium Control
O A
5 4
0/5 4/4
0/5 0/4
0/5 0/4
0/5 0/4
* D a t a e x p r e s s e d as n u m b e r p o s i t i v e o v e r n u m b e r tested.
arized corneas; because of the technical difficulties of detecting the fragile ABO antigens; and because the question of whether one of the major human transplan tation antigen systems, the ABO sys tem, is represented on corneal endotheli um is crucial to the understanding of which antigenic determinants on corneal endothelium stimulate an immune re sponse. Using the indirect immunofluorescence technique, Herold 2 showed blood group antigens A and B on human corneal epithelium, but could not detect these antigens on Bowman's membrane, stroma, Descemet's membrane, or endo thelium. We have now confirmed these findings by using direct immunofluorescence and the ultrasensitive mixed agglu tination reaction, two separate techniques, which are different from the technique used by Herold. Szulman 12 pointed out that A antigen deteriorates from a speci men within three to six hours if left in situ (as, for example, awaiting autopsy) and within 12 hours if stored at 4°C (as a surgical specimen, for example). One might argue that Herold failed to detect ABO antigens on corneal endothelium because he used eye bank eyes up to ten hours after death. Our tissue was process ed fresh or was frozen at — KfC, and the results were identical to those of Herold. We believe that the ABO blood group antigens can play no direct role in specific endothelial rejection, because they are not present on human corneal endothelium.
VOL. 87, NO. 2
LACK OF BLOOD GROUP ANTIGEN A
169
Fig. 2 (Foster and Allansmith). Top left, Corneal endothelial cells from blood type A patient. Slide has been treated with anti-A antiserum followed by washing and application of blood type A human red blood cells. Note lack of adsorption of red blood cells onto endothelial cell membranes. Top right, Corneal epithelial cells from blood type A patient. Slide has been treated with anti-A antiserum followed by washing and application of blood type A human red blood cells. Note dense pack and clustering of red blood cells in rosette formation around the epithelial cells. Bottom left, Corneal epithelial cells from blood type A patient. Slide has been treated with control antiserum followed by washing and application of blood type A human red blood cells. Note lack of adsorption of red blood cells onto epithelial cell membranes. Identical picture obtained if these epithelial cells were treated with anti-A antiserum followed by washing and application of blood type B or blood type 0 red blood cells. Bottom right, Corneal endothelial cells from blood type 0 patient. Slide has been treated with anti-A antiserum, followed by washing and application of blood type A human red blood cells. Note lack of adsorption of red blood cells onto endothelial cell membranes. Identical pictures obtained when blood type O and blood type B indicator red blood cells were used and when control antiserum was used (phase contrast microscopy, x500).
170
AMERICAN JOURNAL OF OPHTHALMOLOGY SUMMARY
Using direct immunofluorescence and mixed agglutination, we did not find blood group antigen A on human corneal endothelium from patients of blood type A or AB. The antigen was easily detected on the epithelium of these patients. We believe that the ABO blood group anti gens are probably absent on human cor neal endothelium, therefore they cannot play a direct role in specific corneal endothelial rejection. REFERENCES 1. Nelken, E., Nelken, D., Michaelson, I. C , and Gurevitch, J.: ABO antigens in the human cornea. Nature 177:840, 1956. 2. Herold, W.: Zum nachweis der blutgrappenantigene A und B in der menschlichen hornhaut mittels der immunofluoreszenztechnik. Klin. Monatsbl. Augenheilkd. 161:658, 1972. 3. Mailath, L., Stenszky, E., Albert, B., and Aszodi, L.: Compatibility examinations of donor's and recipient's blood group in connection with keratoplasty. Debrecen. Szemeszet. 109:112, 1972. 4. Bushmich, D. C , and Nikulina, N. B.: The
FEBRUARY, 1979
significance for the results of keratoplasty of select ing donor recipient pair according to group, type and rhesus characteristics. Oftalmol. Zh. 27:528, 1972. 5. Meyer, H. J.: The importance of blood factors in keratoplasty. Klin. Monatsbl. Augenheilkd. 158: 780, 1971. 6. Allansmith, M. R., Drell, D. W., Kajiyama, C , and Fine, M.: ABO blood groups and corneal trans plantation. Am. J. Ophthalmol. 79:493, 1975. 7. Batchelor, J. R., Casey, T. A., Gibbs, D. C , Lloyd, D. F., Werb, A., Prasad, S. S., and Jones, A.: HLA matching and corneal grafting. Lancet 1:551, 1976. 8. Landsteiner, K.: Uber agglutinationserscheinungen normalen menschlichen blutes. Klin. Wochenscher. 14:1132, 1901. 9. Coombs, R. R. A., Bedford, D., and Rouillard, L. M.: A and B blood group antigens on human epidermal cells demonstrated by mixed agglutina tion. Lancet 1:461, 1956. 10. Hogman, C. F.: Blood group antigens A and B determined by means of mixed agglutination on cultured cells of human fetal kidney, liver, spleen, lung, heart and skin. Vox. Sang. 4:319, 1959. 11. : The principle of mixed agglutination applied to tissue culture systems. Vox. Sang. 4:12, 1959. 12. Szulman, A. E.: The histological distribution of blood group substance A and B in man. J. Exp. Med. 111:785, 1960.
