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Detection of clinically relevant antibodies pretransplant and posttransplant with PRA-STAT
Sirolimus
ELSEVIER
Detection of Clinically Relevant Antibodies Pretransplant and Posttransplant With PRA-STAT J. Neylan, W. de Smet, B. Stijlemans, anti-CD45 study group
K. Brinker, T. Gonwa, J. Moran, and A. Segers on behalf of the
P
RETRANSPLANT and posttransplant (TX) follow-up of transplant patients is done by complement-dependent cytotoxicity (CDC) or flow cytometry assays. As an alternative to these laboratory techniques, an enzymelinked immunoassay (ELISA, PRA-STAT) was recently introduced. PRA-STAT makes use of 46 different soluble HLA preparations for the detection of both complement and noncomplement !?xing HLA-specific IgG. The clinical relevance of the latter method has recently been suggested by several studies.le5 Percentage panel reactive antibody (% PRA) values determined by ELISA in pre-Tx serum samples correlate with the occurrence of post-TX rejection episodes and graft loss in kidney transplant patients.le4 The same holds true for cardiac allograft recipients. Those with pre-Tx PRA higher than 10% are at a three times higher risk for early rejection and have a higher incidence of developing post-TX coronary artery disease.5 In addition, preliminary results indicate that an increase in % PRA post-TX is associated with the occurrence of rejection episodes in kidney transplant patients.4 Such a correlation with clinical events post-TX has3 or has not6 always been found with the more conventional methods as CDC. In view of these findings, we evaluated the use of PRA-STAT as a pre-Tx and post-TX monitoring tool.
MATERIALS AND METHODS Patients and Samples A retrospective ELISA analysis was performed on 256 sera from 64 patients transplanted with kidneys from cadaveric donors. For each
0041-l 315/97/$17.00 PII SOO41-1345(96)00289-8
Table 1. Correlation Between Graft Loss and Presence of Mismatch-Specific Antibodies in the Crossmatch Serum as Detected by PRA-STAT Mismatch-specific antibodies present in crossmatch serum Graft loss due to rejection
Yes
Yes
2
No 1
No
1
60
P = .0043, Fisher Exact Test.
recipient, one pre-Tx (crossmatch serum) and three post-TX (days 2, 15, and 30) sera were tested. All recipients were transplanted across a negative CDC crossmatch. Final crossmatch methods were center dependent and included standard National Institutes of Health CDC and anti-human globulin. Rejection episodes were clinically diagnosed and confirmed by biopsies. The samples were obtained from a multicenter placebo-controlled trial studying drug effectiveness for prevention of acute graft rejection. PRA-STAT
ELISA
The test was performed according to the instructions of the manufacturer (SangStat). Briefly, 100 ~1 of diluted (1901 with specimen-conjugate diluent) patient serum samples was put on microtiter wells coated with an anti-HLA class I capturing monoclonal antibody. After an incubation of 2 hours at room temperaFrom Emory University Hospital, Atlanta, Georgia; Nextran, Brussels, Belgium; Methodist Medical Center, Baylor University Medical Center, Dallas, Texas; and Baxter Healthcare, Chicago, Illinois. Supported by grant. Address reprint requests to Walter De Smet, Sane Stat Medical Corp, 1505 Adams Drive, Menlo Park, CA 94025.
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
330
Transplantation Proceedings, 29, 330-332
(1997)
PRA-STAT DETECTION OF ANTIBODIES
3
6
7
3
11
13
16
331
17
I9
21
23
25
27
29
31
33
36
37
39
,,
43
45
,I
Fig 1. Antibody profile for four consecutive serum samples (crossmatch serum, day 2, day 15, and day 30 after transplantation) of a transplant patient who had no immunologic complications. There is no change in antibody pattern (neither in the % PRA nor in the antibody concentration or affinity). Delta optical densities (ie, after subtraction of the background optical density in the no antigen well) are plotted on the y-axis versus well number on the x-axis. The dashed horizontal line at an optical density of 0.413 represents the cutoff between positive and negative wells.
ture, the plates were washed three times with wash buffer by means of an automatic Ultrawash Plus ELBA plate washer (Dynatech). The wells were then incubated with 100 PL of appropriately diluted peroxidase-labeled rabbit anti-human IgG for 1 hour at room temperature and washed again (as described above). Finally, the wells were incubated with substrate (0.5 mg/mL o-phenylene diamine dihydrochloride) for 15 minutes in the dark, after which the colour reaction was stopped by adding 100 PL of stop solution (1 N hydrochloric acid). Optical densities were read at 490 nm with a reference wavelength of 630 nm (MR-5000 reader, Dynatech).
Post-TX
Rise in Percentage
Panel Reactive
Antibody
A significant correlation (P = .0097, Fisher exact test) between post-TX increase in PF&4 of at least 10% (comparison of PRA value on days 15 and/or 30 with the pre-Tx value) and incidence of biopsy-proven rejection episodes during the first 3 months post-TX was observed (results not shown). Patients showing a post-TX increase in PRA of at least 10% had an eightfold greater risk for acute graft rejection than patients without such an increase. These data confirm the recent results from Monteiro et aL4
RESULTS Graft Loss Antibody
Three patients of the 64 studied lost their graft in the first month due to acute graft rejection (Table 1). In two of these three, antibodies against HLA specificities of the mismatch could clearly be demonstrated in the crossmatch serum. For the third patient, such antibodies were just below the assay cut-off pre-Tx, but rose to a high titer post-TX. Another patient with mismatch-specific antibodies present pre-Tx experienced a severe rejection but did not suffer from graft loss.
Pattern
In addition to % PRA and antibody specificity, the antibody profile (a graph where the optical density for each different HLA phenotype is plotted) is the third parameter that can be obtained with the PRA-STAT test. Due to the technique’s reproducibility,’ this antibody pattern allows one to carefully monitor the patient’s HLA-specific antibody status over time. This is clearly illustrated in Figs 1 and 2.
% 3
332
3.00
NEYLAN, DE SMET, STIJLEMANS
ET AL
PRA 4 0
lotdone ultdone
2.10
85 83
2.00
3 a =
2 I
d
1.00
B g 1.00
3 0.60
0.00 3
6
7
8
11
13
16
17
1B
21
23
26
27
29
31
33
31
37
38
41
43
46
47
Well Number
Fig 2. Antibody profile for four consecutive serum samples (crossmatch serum, day 2, day 15, and day 30 after transplantation) of a patient who suffered from acute graft rejection on day 6 and had to undergo nephrectomy on day 14. A drastic change in the HLA-specific antibody profile can be observed. The dashed horizontal line at an optical density of 0.447 represents the cutoff between positive and negative wells.
CONCLUSION
PRA-STAT may allow for the detection of mismatchspecific IgG alloantibodies that may not be detected by standard cytotoxicity assays including anti-human globulin technology. PRA-STAT may allow for the identification of patients at high risk for acute graft rejection. Donor-specific changes in PRA-STAT results are highly correlative with the incidence of acute graft rejection and/or immunologic graft loss. Future prospective studies will be useful in determining whether PRA-STAT is an effective tool for pre-Tx and post-TX monitoring of kidney graft recipients.
REFERENCES
1. Kerman RH, Susskind B, Buelow R, et al: Transplantation (in press) 2. Regan J, Monteiro F, Speiser D, et al: Am J Kidney Dis (in press) 3. Hourmant M, Perretto S, Le Mauff B, et al: Human Immunol 47:131, 1996 (abstract) 4. Monteiro F, Mineiro C, Rodrigues H, et al: Human Immunol 47:131, 1996 (abstract) 5. Kerman R, Susskind B, Kerman D, et al: Human Immunol 47:131, 1996 (abstract) 6. Bryan CF, Baier KA, Floria-Ginter G, et al: Transplantation 60:1588, 1995 7. Buelow R, Mercier I, Glanville L, et al: Human Immunol 41:1, 1995
ELSEVIER
Detection of Clinically Relevant Antibodies Pretransplant and Posttransplant With PRA-STAT J. Neylan, W. de Smet, B. Stijlemans, anti-CD45 study group
K. Brinker, T. Gonwa, J. Moran, and A. Segers on behalf of the
P
RETRANSPLANT and posttransplant (TX) follow-up of transplant patients is done by complement-dependent cytotoxicity (CDC) or flow cytometry assays. As an alternative to these laboratory techniques, an enzymelinked immunoassay (ELISA, PRA-STAT) was recently introduced. PRA-STAT makes use of 46 different soluble HLA preparations for the detection of both complement and noncomplement !?xing HLA-specific IgG. The clinical relevance of the latter method has recently been suggested by several studies.le5 Percentage panel reactive antibody (% PRA) values determined by ELISA in pre-Tx serum samples correlate with the occurrence of post-TX rejection episodes and graft loss in kidney transplant patients.le4 The same holds true for cardiac allograft recipients. Those with pre-Tx PRA higher than 10% are at a three times higher risk for early rejection and have a higher incidence of developing post-TX coronary artery disease.5 In addition, preliminary results indicate that an increase in % PRA post-TX is associated with the occurrence of rejection episodes in kidney transplant patients.4 Such a correlation with clinical events post-TX has3 or has not6 always been found with the more conventional methods as CDC. In view of these findings, we evaluated the use of PRA-STAT as a pre-Tx and post-TX monitoring tool.
MATERIALS AND METHODS Patients and Samples A retrospective ELISA analysis was performed on 256 sera from 64 patients transplanted with kidneys from cadaveric donors. For each
0041-l 315/97/$17.00 PII SOO41-1345(96)00289-8
Table 1. Correlation Between Graft Loss and Presence of Mismatch-Specific Antibodies in the Crossmatch Serum as Detected by PRA-STAT Mismatch-specific antibodies present in crossmatch serum Graft loss due to rejection
Yes
Yes
2
No 1
No
1
60
P = .0043, Fisher Exact Test.
recipient, one pre-Tx (crossmatch serum) and three post-TX (days 2, 15, and 30) sera were tested. All recipients were transplanted across a negative CDC crossmatch. Final crossmatch methods were center dependent and included standard National Institutes of Health CDC and anti-human globulin. Rejection episodes were clinically diagnosed and confirmed by biopsies. The samples were obtained from a multicenter placebo-controlled trial studying drug effectiveness for prevention of acute graft rejection. PRA-STAT
ELISA
The test was performed according to the instructions of the manufacturer (SangStat). Briefly, 100 ~1 of diluted (1901 with specimen-conjugate diluent) patient serum samples was put on microtiter wells coated with an anti-HLA class I capturing monoclonal antibody. After an incubation of 2 hours at room temperaFrom Emory University Hospital, Atlanta, Georgia; Nextran, Brussels, Belgium; Methodist Medical Center, Baylor University Medical Center, Dallas, Texas; and Baxter Healthcare, Chicago, Illinois. Supported by grant. Address reprint requests to Walter De Smet, Sane Stat Medical Corp, 1505 Adams Drive, Menlo Park, CA 94025.
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
330
Transplantation Proceedings, 29, 330-332
(1997)
PRA-STAT DETECTION OF ANTIBODIES
3
6
7
3
11
13
16
331
17
I9
21
23
25
27
29
31
33
36
37
39
,,
43
45
,I
Fig 1. Antibody profile for four consecutive serum samples (crossmatch serum, day 2, day 15, and day 30 after transplantation) of a transplant patient who had no immunologic complications. There is no change in antibody pattern (neither in the % PRA nor in the antibody concentration or affinity). Delta optical densities (ie, after subtraction of the background optical density in the no antigen well) are plotted on the y-axis versus well number on the x-axis. The dashed horizontal line at an optical density of 0.413 represents the cutoff between positive and negative wells.
ture, the plates were washed three times with wash buffer by means of an automatic Ultrawash Plus ELBA plate washer (Dynatech). The wells were then incubated with 100 PL of appropriately diluted peroxidase-labeled rabbit anti-human IgG for 1 hour at room temperature and washed again (as described above). Finally, the wells were incubated with substrate (0.5 mg/mL o-phenylene diamine dihydrochloride) for 15 minutes in the dark, after which the colour reaction was stopped by adding 100 PL of stop solution (1 N hydrochloric acid). Optical densities were read at 490 nm with a reference wavelength of 630 nm (MR-5000 reader, Dynatech).
Post-TX
Rise in Percentage
Panel Reactive
Antibody
A significant correlation (P = .0097, Fisher exact test) between post-TX increase in PF&4 of at least 10% (comparison of PRA value on days 15 and/or 30 with the pre-Tx value) and incidence of biopsy-proven rejection episodes during the first 3 months post-TX was observed (results not shown). Patients showing a post-TX increase in PRA of at least 10% had an eightfold greater risk for acute graft rejection than patients without such an increase. These data confirm the recent results from Monteiro et aL4
RESULTS Graft Loss Antibody
Three patients of the 64 studied lost their graft in the first month due to acute graft rejection (Table 1). In two of these three, antibodies against HLA specificities of the mismatch could clearly be demonstrated in the crossmatch serum. For the third patient, such antibodies were just below the assay cut-off pre-Tx, but rose to a high titer post-TX. Another patient with mismatch-specific antibodies present pre-Tx experienced a severe rejection but did not suffer from graft loss.
Pattern
In addition to % PRA and antibody specificity, the antibody profile (a graph where the optical density for each different HLA phenotype is plotted) is the third parameter that can be obtained with the PRA-STAT test. Due to the technique’s reproducibility,’ this antibody pattern allows one to carefully monitor the patient’s HLA-specific antibody status over time. This is clearly illustrated in Figs 1 and 2.
% 3
332
3.00
NEYLAN, DE SMET, STIJLEMANS
ET AL
PRA 4 0
lotdone ultdone
2.10
85 83
2.00
3 a =
2 I
d
1.00
B g 1.00
3 0.60
0.00 3
6
7
8
11
13
16
17
1B
21
23
26
27
29
31
33
31
37
38
41
43
46
47
Well Number
Fig 2. Antibody profile for four consecutive serum samples (crossmatch serum, day 2, day 15, and day 30 after transplantation) of a patient who suffered from acute graft rejection on day 6 and had to undergo nephrectomy on day 14. A drastic change in the HLA-specific antibody profile can be observed. The dashed horizontal line at an optical density of 0.447 represents the cutoff between positive and negative wells.
CONCLUSION
PRA-STAT may allow for the detection of mismatchspecific IgG alloantibodies that may not be detected by standard cytotoxicity assays including anti-human globulin technology. PRA-STAT may allow for the identification of patients at high risk for acute graft rejection. Donor-specific changes in PRA-STAT results are highly correlative with the incidence of acute graft rejection and/or immunologic graft loss. Future prospective studies will be useful in determining whether PRA-STAT is an effective tool for pre-Tx and post-TX monitoring of kidney graft recipients.
REFERENCES
1. Kerman RH, Susskind B, Buelow R, et al: Transplantation (in press) 2. Regan J, Monteiro F, Speiser D, et al: Am J Kidney Dis (in press) 3. Hourmant M, Perretto S, Le Mauff B, et al: Human Immunol 47:131, 1996 (abstract) 4. Monteiro F, Mineiro C, Rodrigues H, et al: Human Immunol 47:131, 1996 (abstract) 5. Kerman R, Susskind B, Kerman D, et al: Human Immunol 47:131, 1996 (abstract) 6. Bryan CF, Baier KA, Floria-Ginter G, et al: Transplantation 60:1588, 1995 7. Buelow R, Mercier I, Glanville L, et al: Human Immunol 41:1, 1995