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HYPERACUTE REJECTION OF KIDNEY ALLOGRAFTS, ASSOCIATED WITH PRE-EXISTING HUMORAL ANTIBODIES AGAINST DONOR CELLS
662
HYPERACUTE REJECTION OF KIDNEY ALLOGRAFTS, ASSOCIATED WITH PRE-EXISTING HUMORAL ANTIBODIES AGAINST DONOR CELLS
Both patients were maintained on peritoneal dialysis for several weeks before transplantation, and during this period the;. received several transfusions of whole blood (table i) beside; 13-5 litres and 7-5 litres of human plasma to replace peritoneal
F. KISSMEYER-NIELSEN
received a cadaver kidney removed immeafter death from patients with fatal head injuries. The donor kidneys were preserved by combined surface cooling and perfusion with ice-cold isotonic glucose solution with added heparin. This procedure, which aims at a rapid cooling dow!: to a range of 0-5 °C has been used in 21 transplantations-I; cadaver kidneys and 4 living-donor kidneys. In all cases excer 3, prompt and brisk diuresis occurred without the necessity of postoperative dialysis. The 3 exceptions were 1 case of reversible ischxmic anuria and the 2 cases of irreversible anuna described here. In these 2 cases renal circulation was re-established after ischæmic periods of 92 and 104 minutes, which are less than our average. The surface temperature of the transplants was 6°C and 5°C just before recirculation. Both kidneys rapidly acquired normal colour, tone, and temperature, and urine secretion was observed within 2 minutes after opening of the anastomoses. Urine secretion continued at a normal rate for about 10 minutes, but then gradually decreased, and completely ceased within 1 hour. Simultaneously the transplanted kidney lost tone and became soft and flabby, and the colour changed to that of a livid, mottled cyanosis. In both cases, the surgical procedure included splenectomy and nephrectomy of the patient’s own kidneys. Postoperativety complete anuria persisted and peritoneal dialysis was resumed. The transplanted kidneys were removed 14 days and 3 davs postoperatively. The patients died 81 and 55 days after transplantation ; in both the cause of death was sepsis and peritonitis.
M.D. Aarhus HEAD OF THE BLOOD BANK AND BLOOD GROUPING LABORATORY, AARHUS KOMMUNEHOSPITAL, AARHUS, DENMARK
STEEN OLSEN Copenhagen
M.D. PROFESSOR OF
PATHOLOGY,
UNIVERSITY OF AARHUS
V. POSBORG PETERSEN M.D. Aarhus PROFESSOR OF INTERNAL
O.
MEDICINE,
UNIVERSITY OF AARHUS
FJELDBORG
Cand. Med. Aarhus ASSISTANT CHIEF SURGEON, AARHUS AARHUS, DENMARK
KOMMUNEHOSPITAL,
TRANSPLANTATION immunity or allergy is predominantly cellular, and the small lymphocyte is assumed to play the principal role as a mediator of homograft rejection. The part played by humoral antibodies in homograft rejection is uncertain (Stetson 1963) and it is almost impossible to demonstrate circulating antibodies after allotransplantations in man (Govaerts 1964). Leucocyte agglutinins (Walford 1958) and evidence of thrombocyte isoimmunisation (Bosch 1965) have been found after skin allotransplantation, but circulating antibodies which are active against donor cells after kidney allotransplantation have not been described. The presence of the " white graft " which is seen in skin allotransplantation patients preimmunised with previous skin grafts, thrombocytes, or leucocytes (Dausset et al. 1965, Rood et al. 1965) indicates that humoral factors may have a decisive influence on the fate of the graft, at least under special circumstances. The " white graft " is rejected without being vascularised and without cellular infiltration. Some workers have reported very rapid rejection of kidney allotransplants in cases when major ABO-incompatibility existed between recipient and donor (Porter 1963). These acute rejections were presumably related to the presence of ABO-antibodies in the recipient and ABO-antigens in the graft, and the reports do indicate that humoral factors may be of importance in human kidney transplantations. In a series of 21 consecutive human kidney allotransplantations, we have seen 2 hyperacute rejections which we believe were related to pre-existing humoral antibodies active against antigens from the cadaver donors. Methods The 2 cases concerned were nos. 14 and 17 in our series of renal allotransplantations (Kissmeyer-Nielsen, Fjeldborg, Posborg Petersen, and Svejgaard 1966). The recipients were both females, aged 33 and 38, in terminal urxmia due to chronic pyelonephritis and chronic glomerulonephritis, respectively. TABLE I-PRINCIPAL SEROLOGICAL DATA BEFORE TRANSPLANTATION IN CASES
14 AND 17
protein Both
loss. patients
diately
Serology The principal serological investigations in our series of kidney allotransplantations aimed at the demonstration of antibodies active against erythrocytes, leucocytes, thrombocytes, and kidney extracts before and after transplantation. The methods are described in more detail elsewhere (KissmeverNielsen and Svejgaard 1966). Erythrocyte antibodies were determined by the methods of Kissmeyer-Nielsen (1965). Leucocyte antibodies.-The method of Greenwalt and Polka (1960) was used with a few minor modifications. The sera were inactivated and investigated for the presence of agglutinins, using leucocyte suspensions procured from fresh defibrinated blood after sedimentation of the red blood-cells by addition of gum acacia.
Thrombocyte antibodies.-Inactivated sera were investigated for agglutinins by the method of Dausset and Malinvaud (19M and for complement-fixing antibodies by the sensitive technique of Aster et al. (1964) which is a minor modification of the qualitative technique used by Shulman et al. (1964). The technique, which uses an amount of complement giving oIll:, about 80% haemolysis, was modified so that the degree of hæmolysis could be determined photometrically. A’BeckmanB ’photometer, 50 mm. cuvettes, and addition of Drabkin’ss solution were used; and suitable positive and negative controls and controls for anticomplementarity of serum and amigen were always included. All glassware used was cleaned in 5 potassium dichromate in sulphuric acid. Kidney antibodies.-Inactivated serum was investigated for complement-fixing kidney antibodies by the same method as was used for investigation for thrombocyte antibodies. Kidn-% tissue was obtained from the contralateral kidney or the dene:. removed immediately after the donor kidney. A saline-solute: extract of the kidney was used as antigen. 1 g. of kidney !1SSëe was washed thoroughly in saline solution after comminution until it was free of red blood-cells, and homogenised is.: Warring blender in 10 ml. of saline solution containing l11I sodium azide. The extract was centrifuged at 1000g 1’0:;.. minutes. The supernatant liquid was used as antigen. Exé:’.:::’ from the contralateral donor kidney was used as annger. for investigations of the serum from the respective recipients.
663 Results
Blood-groups of the recipients
TABLE II-THE RESULTS OF SEROLOGICAL INVESTIGATIONS ON FRACTIONS
and donors
OBTAINED BY DEAE CELLULOSE CHROMATOGRAPHY
were:
Cace 14
Recipent.-Ai, CcDee, Fy(a+), K-, Jk(a +), Le(a-b-), 1iBs, Lu(a-), Pl. DoMor.—At, CcDee, Fy(a+), K-, Jk(a+), Le(a-b-), B1s, Lu(a-), P,. Case 17
Recipient.-B, CcDee, Fy(a-), K-, Le(a-b-), MNs, Lu(a-), Pt. DOllor.-B, Cdejcde, Fy(a+), K-, Le(a-b+), NNs, Lu(a--), P,.
irregular erythrocyte antibodies were present in the serum of any of the pairs. The principal serological findings before transplantation in the two recipients are No
summarised in table
i.
shows that both patients were heavily isoimmunised by transfusions and pregnancies. Both patients had extremely strong leucocyte agglutinins with titres above 1/512 and complement-fixing thrombocyte antibody titres of 1/16 and 1/2 respectively. The last column in table i shows the results of investigations performed during and after the operations on pretransplantation sera from the recipients against leucocytes, thrombocytes, and kidney antigens from their respective donors. All the antibodies except the complement-fixing thrombocyte antibodies in case 14 were active against antigens from their respective donors. Unfortunately, leucocyte agglutinins were not titrated against the donor leucocytes, but the reactions obtained with undiluted serum and serum diluted 1/2 and 1/4 were just as strong 2s those obtained with the strongest positive normal leucocyte suspensions. The serum from both recipients contained complement-fixing antibodies when tested against kidney antigen from their respective donors (titre in case 14, 1/32, and in case 17, 1/4). Table
i
Characterisation
of the Antibodies
The various antibodies found
investigated by diethylaminoethyl-cellulose chromatography. The principal results of these investigations are shown in table 11. were
The various antibodies were found in many different fractions, indicating that they were IgG, IgA, besides IgM. The complement-fixing thrombocyte and kidney antibodies were found in almost identical fractions which included both IgG and IgA. The leucocyte agglutinins were found only in the IgG, and IgM fractions.
Specificity
of the Antibodies
The leucocyte agglutinins.-These antibodies reacted with varying strength with leucocytes from many different donors and not at all with leucocytes from some donors. The specificity could not be determined, but the sera undoubtedly contained a mixture of several antibodies.
Absorption experiments were not performed. The complement-fixing thrombocyte antibodies.-The antibodies from case 14 were relatively weak. They were undoubtedly specific since they reacted only with thrombocytes from 4 out of 19 ABO-compatible panel donors. The specificity was unknown. The antibodies from case 17 were fairly strong (1/16), and they had anti-8a specificity. There were strongly positive reactions with ten 8’a--), weakly positive reactions with two 8(a+ ?), and negative reactions with seven 8(a-) platelet suspensions. This gives a P < 0-001 for anti-8a specificity. The complement-fixing kidney antibodies.-The possible ;pecificity of these antibodies was investigated using a group of ABO-compatible kidney antigens. Pre-
transplantation sera were not available for these investigations. Case 14 was investigated using serum taken 6 weeks after transplantation. The antibody reacted positively with four kidney antigens, negatively with seven, and negatively or questionably positively with four antigens. A fortnight later serum reacted positively with all seven kidney antigens including three antigens with which only negative or very questionable reactions had been obtained during the first investigation. Case 17 was investigated 11 days after transplantation. The antibody at that time reacted positive with ten and negative with two kidney antigens. All the kidney antigens used could fix complement with positive control sera. Leucocyte and thrombocyte groupings.-Leucocytes and thrombocytes from the 2 patients and their respective donors were tested against a small number of highly selected sera containing leucocyte agglutinins and/or complement-fixing thrombocyte antibodies. Unfortunately, the specificity of most of our sera is unknown, but it was found that both recipients were 8 (a-), the donor for case 14 was 8(a-), and the donor for case 17 was 8(a+). Other discrepancies between the recipients and their donors were found, but since the specificity of the sera is unknown, this will not be discussed further. Pathological Findings Kidney Lesions Case 14.-The
allograft was removed transplantation. Its weight was 247
2 weeks after the
g.; it measured 13 x 7 x 5 cm. The surface was covered by pus; the cut surface showed total necrotic parenchyma. The cortex
bright red; the medulla of a darker red colour. Only outlines of tubules, vessels, and glomeruli could be identified microscopically. No interstitial-cell infiltration was seen, except a severe granulocyte infiltration in the most was
Fig. 1-Thrombi
of
juxtaglomerular arteriole (case 14).
Phosphotungstic-acid/hasmatoxylin.
664 Discussion The 2 cases described are very similar. Both patients were females in terminal uraemia. They were heavily isoimmunised by pregnancies and previous blood-trans-
fusions, which gave rise to strong circulating antibodies active against blood elements and kidney tissue. Both patients received kidney allografts from cadavers, and the grafts were rejected during the operation, and the rejected kidneys had almost identical microscopical pictures. The circulating antibodies present in both recipients when the transplantations were performed were active against and. gens from the cadaver donors used.
Fig. 2-Macroscopical appearance of the cortical necrosis in the kidney graft in case 17. zone of the outer cortex. The necrotic graft outlined by leucocytes from the recipient. Staining for fibrin by the methods of Weigert and Lendrum and with phosphotungstic-acid/hxmatoxylin demonstrated small thrombi in most glomeruli and juxtaglomerular arterioles (fig. 1). The renal artery distal to the anastomosis and the larger ramifications were occluded by thrombi without any signs of organisation. The walls of these arteries were well preserved and there was no necrosis. Case 17.-This allograft was removed 3 days after transplantation. It measured 12 x 8 x 7 cm. and weighed 239 g. The cut surface revealed a characteristic picture of a total cortical necrosis with a sharp demarcation line in the inner cortex about 1 mm. from the borderline between the cortex and medulla (fig. 2). The parenchyma was deep red, slightly brighter in the cortex. Microscopy revealed total necrosis of the cortex without signs of interstitial infiltration between the remnants of tubules. The necrotic zone was demarcated from an inner, very narrow cortical zone by slight granulocyte infiltration. The medulla was well preserved, but hyperxmic. Staining for fibrin (by the methods specified above) revealed numerous microthromboses in the capillaries of most glomeruli and juxtaglomerular arterioles (fig. 3). Smaller arteries showed necrosis of the wall, generally without leucocyte infiltration. The endothelial cells in small and medium-sized arteries were proliferating, often containing pyroninophilic substance in the cytoplasm. The renal artery and vein were patent in all places.
peripheral was
Fig. 3-Microthrombi in glomeruli and small arterioles in kidney graft of
case
17.
Similar cases have not been described previously. Clinical hyperacute rejections have been seen when major ABO-incompatibility existed between recipient and donor (Porter 1963), but no reports mention the combination of severe isoimmunisation, strong isoantibodies active against donor antigens, hyperacute rejection, and a very characteristic microscopic picture. The findings in the 2 cases described here are of importance in relation to some of the problems in transplantation allergy, and will be discussed in some detail. Both grafts were rejected with extensive glomerular microthromboses without any cellular infiltration. This strongly indicates that humoral antibodies active against antigens in the graft may have a decisive effect on the fate of the graft. This is in disagreement with the generally allergy is at least preaccepted view that transplantation " of cell-fixed and dominantly delayed sensitivity " type. The 2 case-histories also indicate that isoimmunisation before the transplantation with blood-transfusions and or pregnancies may be of very great importance; this is in agreement with the analysis of our whole series of kidney allotransplants (Kissmeyer-Nielsen et el. 1966). Since leucocytes and thrombocytes are of importance in these isoimmunisations, and since both patients also had complement-fixing antibodies against kidney antigen, our results strongly suggest that leucocytes and thrombocytes contain transplantation antigens. This is in agreement with the results of other experiments in which human skin-grafts were used (Bosch 1965, Dausset et al. 1965, Rood et al. 1965). It might be argued that the positive results obtained with kidney antigen and complementfixation technique were caused by contamination of the kidneys with leucocytes and/or thrombocytes. But leucocytes treated in the same way as the kidney antigen give negative results when tested against complement-fixing leucocyte antibodies. Furthermore, the kidney tissue used was thoroughly washed, and the contamination with thrombocytes and leucocytes would have to be fairly heavy to account for the complement fixation obtained. In both cases, the microscopic picture was dominated by widespread microthrombi in the glomeruli and juxtaglomerular arterioles. Typical cortical necrosis was noted in the transplanted kidney of case 17. It is well known that renal cortical necrosis is accompanied by, and probably caused by, microthrombi in the glomeruli, presenting a picture very similar to that seen in the experimental Shwartzman reaction (Hjort and Rapaport 1965, McKay 1965) (fig. 4’. Case 14 also revealed numerous microthrombi, but corticai necrosis could not be demonstrated because of a ion! necrosis developing during the long period in which the non-functioning graft remained in the recipient before the graft was removed. It must be assumed that the isoantibodies present tC
I ,
I’
!
665 There
many similarities between the gross and appearance of our kidney grafts and the kid-
are
microscopic
cases with generalised Shwartzman reaction followed by bilateral cortical necrosis (McKay 1965). Clearly, the glomerular microthrombi in our rejected grafts were not part of a real generalised Shwartzman reaction since the " experimental design " was quite different. The similarity might be explained by the very special situation which is found in kidney allotransplantation in presensitised recipients. The allotransplant is a local antigen challenge to the sensitised recipient, and this is more similar to the situation in the Arthus’ or local Shwartzman reactions than to that giving a generalised Shwartzman reaction.
neys from
Summary Two cadaver kidney allografts underwent hyperacute rejections with extensive glomerular microthromboses and a microscopic picture almost identical to that seen in renal cortical necrosis after the generalised Schwartzman reaction. Both patients were heavily isoimmunised by bloodtransfusions and pregnancies, and before the transplantations their serum contained complement-fixing thrombocyte and kidney antibodies besides leucocyte agglutinins. The antibodies were active against antigens from the cadaver donors used. It is believed that these humoral antibodies played a decisive part in the hyperacute rejections, which happened within 1 hour of the re-establishment of renal circulation and initial urine flow. Fig. 4-Microthrombi in glomeruli in:
(A) kidney graft in case 17; and (B) rabbit with experimental generalised Shwartzman reaction. the recipient were active against antigens in or at the endothelium of the glomeruli and juxtaglomerular arterioles of the grafts. It is reasonable to assume that the reaction between these antibodies and graft antigens functioned as a trigger mechanism in the formation of microthrombi leading to impairment of the cortical bloodflow and subsequent total cortical necrosis. It might be postulated that the rejection was due to a reaction between recipient antibodies and donor leucocytes and thrombocytes present in the grafts and not to a direct reaction with donor-kidney antigens. But this is not feasible since both grafts were thoroughly perfused
before transplantation. There can be little doubt that a very acute rejection, analogous to a second-set rejection, happened in the 2 cases reported. The clinical course and the serological and pathological findings indicate that a specific transplantation allergy existed in the 2 patients, and that humoral antibodies played a predominant part in this allergy. This does not imply that the more common transplantation allergy (delayed sensitivity) did not also exist, but the humoral antibodies must be assumed to have destroyed the graft beford the delayed reaction had time to develop since no infiltration with lymphocytes was found. The only known type of allograft rejection which has some clinical similarity to that described here is the " white graft " seen in experimental skin grafting in presensitised individuals. The grafting of kidneys implies the establishment of abundant blood-supply from host to graft and consequently excellent conditions for humoral, pre-existing antibodies to react with foreign antigens in the graft, and only in the
g.
We thank Lisbeth Kjerbye and Søren Glavind-Kristensen for technical assistance. This work was aided by grants from the Danish State Research Foundation, the King Christian X Foundation, and the Daell Foundation. Requests for reprints should be addressed to F. K.-N., Aarhus Kommunehospital, Aarhus, Denmark. REFERENCES
Aster, R. H., Cooper, H. E., Singer, D. L. (1964) J. Lab. clin. Med. 57, 161. Bosch, L. J. (1965) in Studies on Platelet Transfusion in Man. Thesis; p. 37. Groningen. Dausset, J., Malinvaud, G. (1954) Sang, 25, 847. Rapaport, F. T., Ivanyi, P., Colombani, J. (1965) in Histocompatibility Testing; p. 63. Copenhagen. Govaerts, A. (1964) Immunological Methods; p. 225. Oxford. Greenwalt, T. J., Polka, R. (1960) Am. J. clin. Path. 33, 358. Hjort, P. F., Rapaport, S. I. (1965) Ann. Rev. Med. 16, 135. Kissmeyer-Nielsen, F. (1965) Scand. J. Hœmat. 2, 331. Fjeldborg, O., Posborg Petersen, V., Svejgaard, A. (1966) Ugeskr. Lœger, 128, 757. Svejgaard, A. (1966) Vox sang. (in the press). McKay, D. C. (1965) Disseminated Intravascular Coagulation. New York —
—
—
and London.
Porter, K. A. (1963) Br. med. Bull. 21, 171. Rood, J. J. van, Leeuwen, A. van, Schippers, A. M. J., Vooys, W. H., Frederiks, E., Balner, H., Eernisse, J. G. (1965) in Histocompatibility Testing; p. 37. Copenhagen. Shulman, N. R., Marder, V. J., Hiller, M. C., Collier, E. M. (1964) Prog. Hemat. 4, 222. Stetson, C. A. (1963) Adv. Immun. 3, 97. Walford, R. L. (1958) Transplant. Bull. 5, 55.
"... I sometimes feel that in the local health authority service we can become so taken up with administering services that we lose sight of the prime object of most of these services: the prevention of disease. For example, we spend a great deal of time, energy and money on infant welfare clinics. The cause for which they were founded has been won, and we should ask ourselves whether these resources could not be better employed on other things. I would like to see the family doctor fulfilling his role as doctor to the family and carrying out the medical aspects of this work for the children on his list. The health visitor should assist him in this, and then be free to organise the health education for the whole family. There is a far greater need for preventive services of this kind for the elderly."-Dr. J. A. G. WATSON, M.O.H.’s annual report for East Sussex for 1965; p. 2.
HYPERACUTE REJECTION OF KIDNEY ALLOGRAFTS, ASSOCIATED WITH PRE-EXISTING HUMORAL ANTIBODIES AGAINST DONOR CELLS
Both patients were maintained on peritoneal dialysis for several weeks before transplantation, and during this period the;. received several transfusions of whole blood (table i) beside; 13-5 litres and 7-5 litres of human plasma to replace peritoneal
F. KISSMEYER-NIELSEN
received a cadaver kidney removed immeafter death from patients with fatal head injuries. The donor kidneys were preserved by combined surface cooling and perfusion with ice-cold isotonic glucose solution with added heparin. This procedure, which aims at a rapid cooling dow!: to a range of 0-5 °C has been used in 21 transplantations-I; cadaver kidneys and 4 living-donor kidneys. In all cases excer 3, prompt and brisk diuresis occurred without the necessity of postoperative dialysis. The 3 exceptions were 1 case of reversible ischxmic anuria and the 2 cases of irreversible anuna described here. In these 2 cases renal circulation was re-established after ischæmic periods of 92 and 104 minutes, which are less than our average. The surface temperature of the transplants was 6°C and 5°C just before recirculation. Both kidneys rapidly acquired normal colour, tone, and temperature, and urine secretion was observed within 2 minutes after opening of the anastomoses. Urine secretion continued at a normal rate for about 10 minutes, but then gradually decreased, and completely ceased within 1 hour. Simultaneously the transplanted kidney lost tone and became soft and flabby, and the colour changed to that of a livid, mottled cyanosis. In both cases, the surgical procedure included splenectomy and nephrectomy of the patient’s own kidneys. Postoperativety complete anuria persisted and peritoneal dialysis was resumed. The transplanted kidneys were removed 14 days and 3 davs postoperatively. The patients died 81 and 55 days after transplantation ; in both the cause of death was sepsis and peritonitis.
M.D. Aarhus HEAD OF THE BLOOD BANK AND BLOOD GROUPING LABORATORY, AARHUS KOMMUNEHOSPITAL, AARHUS, DENMARK
STEEN OLSEN Copenhagen
M.D. PROFESSOR OF
PATHOLOGY,
UNIVERSITY OF AARHUS
V. POSBORG PETERSEN M.D. Aarhus PROFESSOR OF INTERNAL
O.
MEDICINE,
UNIVERSITY OF AARHUS
FJELDBORG
Cand. Med. Aarhus ASSISTANT CHIEF SURGEON, AARHUS AARHUS, DENMARK
KOMMUNEHOSPITAL,
TRANSPLANTATION immunity or allergy is predominantly cellular, and the small lymphocyte is assumed to play the principal role as a mediator of homograft rejection. The part played by humoral antibodies in homograft rejection is uncertain (Stetson 1963) and it is almost impossible to demonstrate circulating antibodies after allotransplantations in man (Govaerts 1964). Leucocyte agglutinins (Walford 1958) and evidence of thrombocyte isoimmunisation (Bosch 1965) have been found after skin allotransplantation, but circulating antibodies which are active against donor cells after kidney allotransplantation have not been described. The presence of the " white graft " which is seen in skin allotransplantation patients preimmunised with previous skin grafts, thrombocytes, or leucocytes (Dausset et al. 1965, Rood et al. 1965) indicates that humoral factors may have a decisive influence on the fate of the graft, at least under special circumstances. The " white graft " is rejected without being vascularised and without cellular infiltration. Some workers have reported very rapid rejection of kidney allotransplants in cases when major ABO-incompatibility existed between recipient and donor (Porter 1963). These acute rejections were presumably related to the presence of ABO-antibodies in the recipient and ABO-antigens in the graft, and the reports do indicate that humoral factors may be of importance in human kidney transplantations. In a series of 21 consecutive human kidney allotransplantations, we have seen 2 hyperacute rejections which we believe were related to pre-existing humoral antibodies active against antigens from the cadaver donors. Methods The 2 cases concerned were nos. 14 and 17 in our series of renal allotransplantations (Kissmeyer-Nielsen, Fjeldborg, Posborg Petersen, and Svejgaard 1966). The recipients were both females, aged 33 and 38, in terminal urxmia due to chronic pyelonephritis and chronic glomerulonephritis, respectively. TABLE I-PRINCIPAL SEROLOGICAL DATA BEFORE TRANSPLANTATION IN CASES
14 AND 17
protein Both
loss. patients
diately
Serology The principal serological investigations in our series of kidney allotransplantations aimed at the demonstration of antibodies active against erythrocytes, leucocytes, thrombocytes, and kidney extracts before and after transplantation. The methods are described in more detail elsewhere (KissmeverNielsen and Svejgaard 1966). Erythrocyte antibodies were determined by the methods of Kissmeyer-Nielsen (1965). Leucocyte antibodies.-The method of Greenwalt and Polka (1960) was used with a few minor modifications. The sera were inactivated and investigated for the presence of agglutinins, using leucocyte suspensions procured from fresh defibrinated blood after sedimentation of the red blood-cells by addition of gum acacia.
Thrombocyte antibodies.-Inactivated sera were investigated for agglutinins by the method of Dausset and Malinvaud (19M and for complement-fixing antibodies by the sensitive technique of Aster et al. (1964) which is a minor modification of the qualitative technique used by Shulman et al. (1964). The technique, which uses an amount of complement giving oIll:, about 80% haemolysis, was modified so that the degree of hæmolysis could be determined photometrically. A’BeckmanB ’photometer, 50 mm. cuvettes, and addition of Drabkin’ss solution were used; and suitable positive and negative controls and controls for anticomplementarity of serum and amigen were always included. All glassware used was cleaned in 5 potassium dichromate in sulphuric acid. Kidney antibodies.-Inactivated serum was investigated for complement-fixing kidney antibodies by the same method as was used for investigation for thrombocyte antibodies. Kidn-% tissue was obtained from the contralateral kidney or the dene:. removed immediately after the donor kidney. A saline-solute: extract of the kidney was used as antigen. 1 g. of kidney !1SSëe was washed thoroughly in saline solution after comminution until it was free of red blood-cells, and homogenised is.: Warring blender in 10 ml. of saline solution containing l11I sodium azide. The extract was centrifuged at 1000g 1’0:;.. minutes. The supernatant liquid was used as antigen. Exé:’.:::’ from the contralateral donor kidney was used as annger. for investigations of the serum from the respective recipients.
663 Results
Blood-groups of the recipients
TABLE II-THE RESULTS OF SEROLOGICAL INVESTIGATIONS ON FRACTIONS
and donors
OBTAINED BY DEAE CELLULOSE CHROMATOGRAPHY
were:
Cace 14
Recipent.-Ai, CcDee, Fy(a+), K-, Jk(a +), Le(a-b-), 1iBs, Lu(a-), Pl. DoMor.—At, CcDee, Fy(a+), K-, Jk(a+), Le(a-b-), B1s, Lu(a-), P,. Case 17
Recipient.-B, CcDee, Fy(a-), K-, Le(a-b-), MNs, Lu(a-), Pt. DOllor.-B, Cdejcde, Fy(a+), K-, Le(a-b+), NNs, Lu(a--), P,.
irregular erythrocyte antibodies were present in the serum of any of the pairs. The principal serological findings before transplantation in the two recipients are No
summarised in table
i.
shows that both patients were heavily isoimmunised by transfusions and pregnancies. Both patients had extremely strong leucocyte agglutinins with titres above 1/512 and complement-fixing thrombocyte antibody titres of 1/16 and 1/2 respectively. The last column in table i shows the results of investigations performed during and after the operations on pretransplantation sera from the recipients against leucocytes, thrombocytes, and kidney antigens from their respective donors. All the antibodies except the complement-fixing thrombocyte antibodies in case 14 were active against antigens from their respective donors. Unfortunately, leucocyte agglutinins were not titrated against the donor leucocytes, but the reactions obtained with undiluted serum and serum diluted 1/2 and 1/4 were just as strong 2s those obtained with the strongest positive normal leucocyte suspensions. The serum from both recipients contained complement-fixing antibodies when tested against kidney antigen from their respective donors (titre in case 14, 1/32, and in case 17, 1/4). Table
i
Characterisation
of the Antibodies
The various antibodies found
investigated by diethylaminoethyl-cellulose chromatography. The principal results of these investigations are shown in table 11. were
The various antibodies were found in many different fractions, indicating that they were IgG, IgA, besides IgM. The complement-fixing thrombocyte and kidney antibodies were found in almost identical fractions which included both IgG and IgA. The leucocyte agglutinins were found only in the IgG, and IgM fractions.
Specificity
of the Antibodies
The leucocyte agglutinins.-These antibodies reacted with varying strength with leucocytes from many different donors and not at all with leucocytes from some donors. The specificity could not be determined, but the sera undoubtedly contained a mixture of several antibodies.
Absorption experiments were not performed. The complement-fixing thrombocyte antibodies.-The antibodies from case 14 were relatively weak. They were undoubtedly specific since they reacted only with thrombocytes from 4 out of 19 ABO-compatible panel donors. The specificity was unknown. The antibodies from case 17 were fairly strong (1/16), and they had anti-8a specificity. There were strongly positive reactions with ten 8’a--), weakly positive reactions with two 8(a+ ?), and negative reactions with seven 8(a-) platelet suspensions. This gives a P < 0-001 for anti-8a specificity. The complement-fixing kidney antibodies.-The possible ;pecificity of these antibodies was investigated using a group of ABO-compatible kidney antigens. Pre-
transplantation sera were not available for these investigations. Case 14 was investigated using serum taken 6 weeks after transplantation. The antibody reacted positively with four kidney antigens, negatively with seven, and negatively or questionably positively with four antigens. A fortnight later serum reacted positively with all seven kidney antigens including three antigens with which only negative or very questionable reactions had been obtained during the first investigation. Case 17 was investigated 11 days after transplantation. The antibody at that time reacted positive with ten and negative with two kidney antigens. All the kidney antigens used could fix complement with positive control sera. Leucocyte and thrombocyte groupings.-Leucocytes and thrombocytes from the 2 patients and their respective donors were tested against a small number of highly selected sera containing leucocyte agglutinins and/or complement-fixing thrombocyte antibodies. Unfortunately, the specificity of most of our sera is unknown, but it was found that both recipients were 8 (a-), the donor for case 14 was 8(a-), and the donor for case 17 was 8(a+). Other discrepancies between the recipients and their donors were found, but since the specificity of the sera is unknown, this will not be discussed further. Pathological Findings Kidney Lesions Case 14.-The
allograft was removed transplantation. Its weight was 247
2 weeks after the
g.; it measured 13 x 7 x 5 cm. The surface was covered by pus; the cut surface showed total necrotic parenchyma. The cortex
bright red; the medulla of a darker red colour. Only outlines of tubules, vessels, and glomeruli could be identified microscopically. No interstitial-cell infiltration was seen, except a severe granulocyte infiltration in the most was
Fig. 1-Thrombi
of
juxtaglomerular arteriole (case 14).
Phosphotungstic-acid/hasmatoxylin.
664 Discussion The 2 cases described are very similar. Both patients were females in terminal uraemia. They were heavily isoimmunised by pregnancies and previous blood-trans-
fusions, which gave rise to strong circulating antibodies active against blood elements and kidney tissue. Both patients received kidney allografts from cadavers, and the grafts were rejected during the operation, and the rejected kidneys had almost identical microscopical pictures. The circulating antibodies present in both recipients when the transplantations were performed were active against and. gens from the cadaver donors used.
Fig. 2-Macroscopical appearance of the cortical necrosis in the kidney graft in case 17. zone of the outer cortex. The necrotic graft outlined by leucocytes from the recipient. Staining for fibrin by the methods of Weigert and Lendrum and with phosphotungstic-acid/hxmatoxylin demonstrated small thrombi in most glomeruli and juxtaglomerular arterioles (fig. 1). The renal artery distal to the anastomosis and the larger ramifications were occluded by thrombi without any signs of organisation. The walls of these arteries were well preserved and there was no necrosis. Case 17.-This allograft was removed 3 days after transplantation. It measured 12 x 8 x 7 cm. and weighed 239 g. The cut surface revealed a characteristic picture of a total cortical necrosis with a sharp demarcation line in the inner cortex about 1 mm. from the borderline between the cortex and medulla (fig. 2). The parenchyma was deep red, slightly brighter in the cortex. Microscopy revealed total necrosis of the cortex without signs of interstitial infiltration between the remnants of tubules. The necrotic zone was demarcated from an inner, very narrow cortical zone by slight granulocyte infiltration. The medulla was well preserved, but hyperxmic. Staining for fibrin (by the methods specified above) revealed numerous microthromboses in the capillaries of most glomeruli and juxtaglomerular arterioles (fig. 3). Smaller arteries showed necrosis of the wall, generally without leucocyte infiltration. The endothelial cells in small and medium-sized arteries were proliferating, often containing pyroninophilic substance in the cytoplasm. The renal artery and vein were patent in all places.
peripheral was
Fig. 3-Microthrombi in glomeruli and small arterioles in kidney graft of
case
17.
Similar cases have not been described previously. Clinical hyperacute rejections have been seen when major ABO-incompatibility existed between recipient and donor (Porter 1963), but no reports mention the combination of severe isoimmunisation, strong isoantibodies active against donor antigens, hyperacute rejection, and a very characteristic microscopic picture. The findings in the 2 cases described here are of importance in relation to some of the problems in transplantation allergy, and will be discussed in some detail. Both grafts were rejected with extensive glomerular microthromboses without any cellular infiltration. This strongly indicates that humoral antibodies active against antigens in the graft may have a decisive effect on the fate of the graft. This is in disagreement with the generally allergy is at least preaccepted view that transplantation " of cell-fixed and dominantly delayed sensitivity " type. The 2 case-histories also indicate that isoimmunisation before the transplantation with blood-transfusions and or pregnancies may be of very great importance; this is in agreement with the analysis of our whole series of kidney allotransplants (Kissmeyer-Nielsen et el. 1966). Since leucocytes and thrombocytes are of importance in these isoimmunisations, and since both patients also had complement-fixing antibodies against kidney antigen, our results strongly suggest that leucocytes and thrombocytes contain transplantation antigens. This is in agreement with the results of other experiments in which human skin-grafts were used (Bosch 1965, Dausset et al. 1965, Rood et al. 1965). It might be argued that the positive results obtained with kidney antigen and complementfixation technique were caused by contamination of the kidneys with leucocytes and/or thrombocytes. But leucocytes treated in the same way as the kidney antigen give negative results when tested against complement-fixing leucocyte antibodies. Furthermore, the kidney tissue used was thoroughly washed, and the contamination with thrombocytes and leucocytes would have to be fairly heavy to account for the complement fixation obtained. In both cases, the microscopic picture was dominated by widespread microthrombi in the glomeruli and juxtaglomerular arterioles. Typical cortical necrosis was noted in the transplanted kidney of case 17. It is well known that renal cortical necrosis is accompanied by, and probably caused by, microthrombi in the glomeruli, presenting a picture very similar to that seen in the experimental Shwartzman reaction (Hjort and Rapaport 1965, McKay 1965) (fig. 4’. Case 14 also revealed numerous microthrombi, but corticai necrosis could not be demonstrated because of a ion! necrosis developing during the long period in which the non-functioning graft remained in the recipient before the graft was removed. It must be assumed that the isoantibodies present tC
I ,
I’
!
665 There
many similarities between the gross and appearance of our kidney grafts and the kid-
are
microscopic
cases with generalised Shwartzman reaction followed by bilateral cortical necrosis (McKay 1965). Clearly, the glomerular microthrombi in our rejected grafts were not part of a real generalised Shwartzman reaction since the " experimental design " was quite different. The similarity might be explained by the very special situation which is found in kidney allotransplantation in presensitised recipients. The allotransplant is a local antigen challenge to the sensitised recipient, and this is more similar to the situation in the Arthus’ or local Shwartzman reactions than to that giving a generalised Shwartzman reaction.
neys from
Summary Two cadaver kidney allografts underwent hyperacute rejections with extensive glomerular microthromboses and a microscopic picture almost identical to that seen in renal cortical necrosis after the generalised Schwartzman reaction. Both patients were heavily isoimmunised by bloodtransfusions and pregnancies, and before the transplantations their serum contained complement-fixing thrombocyte and kidney antibodies besides leucocyte agglutinins. The antibodies were active against antigens from the cadaver donors used. It is believed that these humoral antibodies played a decisive part in the hyperacute rejections, which happened within 1 hour of the re-establishment of renal circulation and initial urine flow. Fig. 4-Microthrombi in glomeruli in:
(A) kidney graft in case 17; and (B) rabbit with experimental generalised Shwartzman reaction. the recipient were active against antigens in or at the endothelium of the glomeruli and juxtaglomerular arterioles of the grafts. It is reasonable to assume that the reaction between these antibodies and graft antigens functioned as a trigger mechanism in the formation of microthrombi leading to impairment of the cortical bloodflow and subsequent total cortical necrosis. It might be postulated that the rejection was due to a reaction between recipient antibodies and donor leucocytes and thrombocytes present in the grafts and not to a direct reaction with donor-kidney antigens. But this is not feasible since both grafts were thoroughly perfused
before transplantation. There can be little doubt that a very acute rejection, analogous to a second-set rejection, happened in the 2 cases reported. The clinical course and the serological and pathological findings indicate that a specific transplantation allergy existed in the 2 patients, and that humoral antibodies played a predominant part in this allergy. This does not imply that the more common transplantation allergy (delayed sensitivity) did not also exist, but the humoral antibodies must be assumed to have destroyed the graft beford the delayed reaction had time to develop since no infiltration with lymphocytes was found. The only known type of allograft rejection which has some clinical similarity to that described here is the " white graft " seen in experimental skin grafting in presensitised individuals. The grafting of kidneys implies the establishment of abundant blood-supply from host to graft and consequently excellent conditions for humoral, pre-existing antibodies to react with foreign antigens in the graft, and only in the
g.
We thank Lisbeth Kjerbye and Søren Glavind-Kristensen for technical assistance. This work was aided by grants from the Danish State Research Foundation, the King Christian X Foundation, and the Daell Foundation. Requests for reprints should be addressed to F. K.-N., Aarhus Kommunehospital, Aarhus, Denmark. REFERENCES
Aster, R. H., Cooper, H. E., Singer, D. L. (1964) J. Lab. clin. Med. 57, 161. Bosch, L. J. (1965) in Studies on Platelet Transfusion in Man. Thesis; p. 37. Groningen. Dausset, J., Malinvaud, G. (1954) Sang, 25, 847. Rapaport, F. T., Ivanyi, P., Colombani, J. (1965) in Histocompatibility Testing; p. 63. Copenhagen. Govaerts, A. (1964) Immunological Methods; p. 225. Oxford. Greenwalt, T. J., Polka, R. (1960) Am. J. clin. Path. 33, 358. Hjort, P. F., Rapaport, S. I. (1965) Ann. Rev. Med. 16, 135. Kissmeyer-Nielsen, F. (1965) Scand. J. Hœmat. 2, 331. Fjeldborg, O., Posborg Petersen, V., Svejgaard, A. (1966) Ugeskr. Lœger, 128, 757. Svejgaard, A. (1966) Vox sang. (in the press). McKay, D. C. (1965) Disseminated Intravascular Coagulation. New York —
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Porter, K. A. (1963) Br. med. Bull. 21, 171. Rood, J. J. van, Leeuwen, A. van, Schippers, A. M. J., Vooys, W. H., Frederiks, E., Balner, H., Eernisse, J. G. (1965) in Histocompatibility Testing; p. 37. Copenhagen. Shulman, N. R., Marder, V. J., Hiller, M. C., Collier, E. M. (1964) Prog. Hemat. 4, 222. Stetson, C. A. (1963) Adv. Immun. 3, 97. Walford, R. L. (1958) Transplant. Bull. 5, 55.
"... I sometimes feel that in the local health authority service we can become so taken up with administering services that we lose sight of the prime object of most of these services: the prevention of disease. For example, we spend a great deal of time, energy and money on infant welfare clinics. The cause for which they were founded has been won, and we should ask ourselves whether these resources could not be better employed on other things. I would like to see the family doctor fulfilling his role as doctor to the family and carrying out the medical aspects of this work for the children on his list. The health visitor should assist him in this, and then be free to organise the health education for the whole family. There is a far greater need for preventive services of this kind for the elderly."-Dr. J. A. G. WATSON, M.O.H.’s annual report for East Sussex for 1965; p. 2.