1095

background colour in any of the 7 c.F. This is probably to be expected, in tested. samples view of the high faecal bulk characteristic of c.F. and hence the high dilution of bile pigments. There

was no

we

RENAL TRANSPLANTATION AND B-CELL CROSS-MATCHES WITH AUTOANTIBODIES AND

ALLOANTIBODIES

Discussion the results discussed are from our laborathe method described is now being used to alone, tory screen a total of 700 newborn babies per week in Auckland, Wellington, and the Waikato and is being established in other main centres. The cost per test (laboratory staff and materials) is 10 c. N.Z. The procedure is very simple, subject to quality control, and precise. The difference in colour between samples from babies with and without c.F. is dramatic. The sample cards are easy to handle for both nursing and laboratory staff. The trypsin activity of dry stool sample is stable for at least several weeks at room temperature. If samples deteriorated, they would appear as false positives, not false negatives as in meconium-albumin screens. We have no evidence that microorganisms could cause false negatives. The quality control we maintain serves as a check on contaminated reagents. It has been reported that 10-15% of children and adolescents with c.F. have normal or near-normal pancreatic function. This has been taken as justification for a significant false-negative rate in neonatal screening tests for C.F.5.11 However, we know of 2 such children who had a documented lack of pancreatic enzyme activity for some months after birth. We believe that there is insufficient information in the literature to enable a reliable estimate to be made of the incidence of normal pancreatic function in newborn babies with c.F. The main cause of false-positive results is probably low enzyme activity due to prematurity. Occasionally we encounter problems due to insufficient sample being applied, or a transitional stool, but this is easily corrected by the nursing staff. Colour masking by bile pigments is a rare cause of false positives. Since stool-trypsin is bound to oc.-macroglobulins, 12 and is hence unable to bind to trypsin inhibitors such as soybean trypsin inhibitor, diet alone would be most unlikely to cause false

positives. We have found by quantitative analysis of stool samples from children that many with trypsin concentrations at the lower end of the normal range may have chymotrypsin concentrations at the upper end of the normal range. We have investigated the possibility of reducing the false-positive rale in screening even further by reassaying positive samples with glutaryl-phenylalanine-p-nitroanilide (G.P.N.A.), a specific substrate for chymotrypsin. However, because of the much lower sensitivity of this assay" we found that the positive-rate was not significantly reduced. With our current positiverate of 0-1%, therefore, we expect to find 1 c.F. in every 3 babies from whom we request a second stool specimen. We are grateful to the many nurses, doctors, and parents who cooperated in this programme. We acknowledge the technical assistance of Mr Kevin Hartfield and many useful discussions with Mr Brian Edgar. The National Child Health Research Foundation and the N.Z. Cystic Fibrosis Association provided financial support.

for reprints should be addressed to J. R. C., Department Pediatrics, School of Medicine, University of Auckland, 85 Park Road, Auckland 3, New Zealand.

Requests

of

P.

J. MORRIS Nuffield Department of Surgery, University of Oxford, Radclijfe Infirmary, Oxford, OX2 6HE A. TING

Although

Of 51 cadaveric kidneys transplanted between June, 1976, and June, 1977, 18 were transplanted in the presence of a positive crossmatch against the donor’s B lymphocytes. 11 of these positive cross-matches were due to alloantibodies and 7 due to autoantibodies. Autoantibodies were defined not only on the basis of autoreactivity with B lymphocytes but also by their absent or restricted reactivity with lymphocytes from patients with chronic lymphocytic leukæmia. Transplants in 8 of 11 patients with a positive alloantibody-B-cell cross-match and in 6 of 7 patients with a positive autoantibody-B-cell cross-match were successful at 3 months. These success-rates were no different from those found in patients with a negative B-cell cross-match. Thus, renal allografts may be performed with a reasonable assurance of success in the presence of a positive B-cell cross-match whether due to autoantibodies or to alloantibodies.

Summary

Introduction HUMAN renal transplantations performed in the preof a positive B-cell cross-match have lately been reported,I-4 and these transplants fail no more often than negatively cross-matched transplants. It has been assumed that these antibodies were directed against allogeneic antigens of the HLA-D-locus-related (D.R.) specificities. However, Park et awl. and ourselves6 have noted that some of these B-cell antibodies react not only with a random panel of allogeneic lymphocytes but also with autologous B lymphocytes. Park and his colleaguess have shown that these autoantibodies are most reactive at 50C (rather than at 20 °C, the usual incubation temperature). We have shown that these autoantibodies are either non-reactive or have restricted reactipatients with chronic vity with B lymphocytes from lymphocytic leukxmia (C.L.L.).66 Park and his colleaguess have implied that successful renal transplants in the presence of a positive B-cell cross-match can be attributed to autoantibodies rather sence

Robinson, P. G., Elliott, R. B., Fraser, J. N.Z. med. J. 1976, 83, 268. Schwachman, H., Redmond, A., Khaw, K.-T., Pediatrics, Springfield, 1970, 46, 335. 3. Elliott, R. B. ibid. 1976, 57, 474. 4. Verdonk, G., Christophe, A. Proc. 9th int. Congr. Nutrition, Mexico, 1972; vol. 1, p. 278. Basel,1975. 5. Prosser, R., Owen, H., Bull, F., Parry, B., Smerkinich, J., Goodwin, H. A., Dathan, J. Archs Dis. Childh. 1974, 49, 597 6. Kollberg, H., Hellsing, K. Acta pœdiat. scand. 1975, 64, 477. 7. Robinson, P. G., Elliott, R. B. N.Z. med. J. 1974, 79, 1024 8. Robinson, P. G., Elliott, R. B. Archs Dis. Childh. 1976, 51, 301 9. Erlanger, B. F., Kokowsky, N., Cohen, W. Archs Biochem. Biophys. 1961, 95, 271. 10. Robinson, P. G., Smith, P. A., Elliott, R. B. Clinica chim. Acta, 1975, 62, 1. 2.

225. 11. Ryley, H. C., Neale, L. M., Brogan, T. D., Bray P. T. ibid. 1975, 64, 117. 12. Haverback, B. J., Dyce, B. J., Gutentag, P. J., Montgomery, D. W. Gas13.

troenterology, 1963, 44, 588. Erlanger, B. F., Edel, F., Cooper, A. G. Archs Biochem. Biophys. 1966, 115, 206.

1096 than alloantibodies against B-lymphocyte antigens. We have examined recipients with a B-cell-positive crossmatch due either to autoantibodies or to alloantibodies. We found no difference in the early failure-rate between the two groups, and therefore we do not agree with the implied conclusions of Park and his colleagues.

TABLE

I1-3

MO TRANSPLANT FUNCTION AND ANTIBODY AND CROSS-MATCH STATUS

Materials and Methods Renal

Transplants

51 cadaveric kidneys were transplanted between June, 1976, and June, 1977. 18 of these transplants were performed in the presence of a positive B-cell cross-match. The clinical details have been described elsewhere.3.. 8 patients had pretransplant antibodies but a negative cross-match, and 24 patients had no pretransplant antibodies. 1 patient was excluded from this analysis because he had pretransplant antibodies that reacted with his donor’s T lymphocytes. Subsequent studies showed that these were autoantibodies. His transplant is functioning 9 months after operation. All patients have been followed up for at least 3 months.

Antibody Screening All sera were screened against a random panel of at least 25 T-cell and corresponding B-cell preparations and B-cells from at least 25 C.L.L. patients. An antibody-positive serum was defined as one reacting with at least two target cells, and reactivity with one target cell was defined as a weak positive (±). A serum reacting with a low frequency against c.L.L. cells was defined as showing restricted C.L.L. reactivity (-).

Cross-matching For cross-matching the serum with the highest frequency of reactions against a random panel was chosen, together with a fresh sample of serum. In the majority of cases cross-matches were performed with the donor’s unseparated splenic lymphocytes and separated T and B cells. In a number of cases where spleen cells were not available, either lymph-node lymphocytes or unseparated peripheral-blood lymphocytes (P.B.L.) were used.3.. The method of Wilson

et

al.

was

TABLE I-REACTIVITY OF CROSS-MATCH SERA

CELLS,

separate T FROM 18 PATIENTS

used

to

and B cells from spleen and peripheral blood. The cytotoxic assay has been described previously.3 Detection ofautoantibodies The sera used for cross-matching were tested against the patients’ autologous unseparated P.B.L. and separated T and B cells. All tests were done in triplicate.

Results Table i summarises the reactivity of the cross-match with autologous and allogeneic T and B cells and with C.L.L. cells. There is a good correlation between autologous reactivity and negative or restricted reactivity with C.L.L. cells. 3 patients (nos. 8, 9, and 10) whose sera were negative with C.L.L. cells failed to react with their own lymphocytes. The sera of these 3 patients had low-frequency B-cell antibodies when tested against a random panel (6% in patient 8, 19% in patient 9, and sera

25% in patient 10). Sera from 6

patients showed

restricted C.L.L. reactiwith autoantibodies. Furthvity, and these were the sera ermore, the sera with autologous antibodies reacted with no more than 6 of the 25 c.L.L.-cell panel. These 6 C.L.L. cells were the same cells and did not share any D.R. antigen in common. A B-cell-positive cross-match in a patient demonWITH SEPARATED T AND B CELLS FROM A RANDOM PANEL, C.L.L.

AND AUTOLOGOUS T AND B CELLS AND HIGHEST REACTIVITY OF THE MOST POSITIVE SERUM FROM EACH PATIENT

I

-L

*s=success, F=failure.

tTechnical failure. +=positive ±=weak positive (-)=restricted

reactivityB

text) (see t

1097 to have autoantibodies was defined as being due these antibodies rather than to alloantibodies. 7 such cases were seen in this study. However, since the sera were not examined by absorption to see whether they also contained alloantibodies we cannot exclude the possibility that such positive cross-matches were due to

strated to

alloantibodies. At 3 months after transplantation 14 of the 18 cases of B-cell-positive cross-matches were successful. 4 grafts failed-3 from rejection and 1 from renal-artery thrombosis. Of the 3 grafts lost from rejection, 2 were second grafts and underwent accelerated rejection within the first week. The 3rd underwent irreversible rejection on

day 40. Also shown in tableare the frequencies of positive reactions of the most positive sera from each patient against a random panel of T and B lymphocytes. The sera of some patients reacted with B lymphocytes only, whereas others reacted with the T and corresponding B lymphocytes of some individuals of the panel as well as the B lymphocytes alone of other panel members. These results show that most of the patients are highly sensitised, 8 patients having reactivity of at least 80%. Furthermore, 2 patients (nos. 6 and 13) had reactivity with 100% of the panel. Patient 13 reacted with 100% of the panel; the reactivity was against both T and B cells of 96% of the panel and against B cells alone of the remaining 4% of the panel. Patient 6 reacted with 100% of the panel B lymphocytes only.

Table ii shows the 3-month transplant function of the patients with (i) B-cell-positive cross-match due to autoantibodies, (ii) B-cell-positive cross-match due to allogeneic antibodies, (iii) lymphocytotoxic antibodies but a negative cross-match, and (iv) no antibodies. 6 of 7 patients (86%) with autologous B-cell antibodies and a positive B-cell cross-match had a successful transplant 3 months. This success-rate was similar to that found in the group with a positive cross-match due to allogeneic antibodies, where 8 of 11 grafts (73%) were successful. This latter group includes one technical failure (patient 11). The results in these two groups of patients are not significantly different from those in the two groups with a negative cross-match, whether they had antibodies (88%) or not (83%). at

cross-match due to autologous B-cell antibodies, and both transplants have been successful. Park et al.1 and ourselves6 have recognised autoantibodies that react predominantly with B cells. Park et all have shown that these autoantibodies react most effectively against a random panel after incubation of serum and cells at 5 1 C. From their studies they suggested that "positive B-lymphocyte crossmatches found in kidney transplantation do not detect alloantibodies". Our study shows that this suggestion is not valid. Of the 18 B-cellpositive cross-matches reported here, 7 were due to autoantibodies and 11 were not. There was 1 rejection failure in the former group, and there were 2 failures in the latter group. There is no apparent reason for these failures. Direct killing of donor lymphocytes by recipient lymphocytes in the absence of antibody was tested in 2 of these 3 instances of rejection and was positive in 1. Thus, pre-existing cellular immunity may have been a factor in determining graft outcome in that patient. Dejelo and Williams8 have suggested, on the basis of their hyperacute rejection with a positive B-cell crossmatch, that "not all positive B-cell crossmatches permit immunologically safe renal allografting". Our results also suggest that some caution should be exercised before transplanting B-cell-positive crossmatches. But it is relevant to note that hyperacute rejections have also been reported in the presence of a negative cross-

match.12 In this study we have no evidence that these B-cell antibodies cause enhancement, as has been demonstrated in the rat.13 However, the practical benefits of transplanting patients with a B-cell-positive cross-match is seen in our study. Of the 18 patients transplanted, 2 had antibodies reacting with 100% of a T and B lymphocyte panel, and 8 patients had reactivity with at least 80% of the panel. These patients are highly sensitised and would thus be considered as either non-transplantable or very difficult to transplant under the present policy of regarding a positive cross-match between donor and recipient as a contraindication to transplantation. We believe, however, that, in general, renal transplantation can be performed safely in the presence of a positive B-cell cross-match, whether due to alloantibodies or to autoantibodies. In most instances the transplant will be successful.

Discussion

Transplants with a positive B-cell cross-match have generally had an early success-rate greater than would have been expected,1-4 although a hyperacute rejection has been reported.8 We have now shown that the graft outcome in patients with a B-cell-positive cross-match is as successful as the group with lymphocytotoxic antibodies but a negative cross-match and the group with no antibodies. All three groups were transplanted within the same period of one year. Autolymphocytotoxic antibodies have been recognised in pretransplant sera .9-11 Cross et al.9 and Stastny and Austin10 did not separate autoantibodies by their reaction with T or B lymphocytes, since in their tests the target cells were unseparated peripheral-blood lymphocytes. Both studies have shown that transplants with a positive cross-match due to autoantibodies have a high success-rate, with no indication of hyperacute rejection. Reekers et al.11 have demonstrated 2 cases of positive

We wish to thank Mr Paul Trotter for his excellent technical assistThis work was supported by a grant from the Medical Research

ance.

Council.

Requests for reprints should be addressed to A.T. REFERENCES 1.

R.

B., Terasaki, P. I., Opelz, G., Malekzadeh, M., Pennisi, A. J., Ettenger, Uittenbogaart, C., Fine, R. Lancet, 1976, ii, 56. 2. Lobo, P. I., Westervelt, R. B., Rudolf, L. E. ibid. 1977, i, 925. 3. Morris, P. J., Ting, A., Oliver, D. O., Bishop, M., Williams, K., Dunnill, M. S. ibid. p. 1288. 4. Morris, P. J., Ting, A., Oliver, D. Transplant. Proc. (in the press). 5. Park, M. S., Terasaki, P. I., Bernoco, D. Lancet, 1977, ii, 465. 6. Ting, A., Morris, P. J. Transplantation (in the press). 7. Wilson, A. B., Haegart, D. G., Coombs, R. R. A. Clin. exp. Immun.

1975, 22,177. 8. Dejelo, C. L., Williams, T. C. Lancet, 1977, ii, 241. 9. Cross, D. E., Greiner, R., Whittier, F. C. Transplantation, 1976, 21, 307. 10. Stastny, P., Austin, C. L. ibid. p. 399. 11. Reekers, P., Lucassen-Hermans, R., Koene, R. A. P., Kunst, V. A. J. M. Lancet, 1977, i, 1063. 12. Morris, P. J., Kincaid-Smith, P., McKenzie, I. F. C., Marshall, V. C., Ting, A. Med. J. Aust. 1969, ii, 379. 13. Soulillou, J. P., Carpenter, C. B.,

1976, 143, 405.

d’Apice,

A.

J., Strom, T.

B.

J.

exp. Med.

Renal transplantation and B-cell cross-matches with autoantibodies and alloantibodies.

1095 background colour in any of the 7 c.F. This is probably to be expected, in tested. samples view of the high faecal bulk characteristic of c.F. a...
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