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Post-transplant monitoring of hematopoietic cell chimerism allows early clinical intervention following bone marrow transplantation
136
Abstracts
7.2 #186
7.2 #187
A COMPARISON OF SENSITIVITY, SPEED, AND RELIABILITY BETWEEN THE SANGSTAT TRANSTAT@AB CARTRIDGE-BASED ENZYME IMMUNOASSAY AND AN ELISA IMMUNOASSAY FOR OKT3 SERUM ANTIBODY DETERMINATIONS. JC Inlow, PK Hennessy, PW Adams, CG Orosz. The Ohio State University Hospitals, Columbus, Ohio. OKT3, an immunosuppressant used to treat acute allograft rejection, in an IgG2a mouse monoclonal antibody directed against the CD3 antigen receptor of the human T lymphocyte. Following treatment with OKT3, some patients develop antibodies against mouse Ig, which may decrease or inhibit the efficacy of monoclonal antibody treatment upon subsequent administration. Two immunoassays used to monitor anti-OKT3 antibody were evaluated: 1) a standard ELISA for human anti-OKT3 IgG, in which patient sera are added to 96 well plates coated with mouse OKT3, and serum antibody binding is detected by enzyme-conjugated rabbit anti-human IgG plus a chromogenic substrate, 2) the Sangstat Transtat®Ab kit, which is a solid phase colorimetric immunoassay in cartridge form. When tested on dilutions of pooled anti-OKT3 positive sera, both methods were equally sensitive and exhibited identical end-point titers of 1 :4000. Next, sera from 21 patients were tested with both methods, resulting in 3 discrepant results. One patient exhibited antibody (anti-idiotypic) detectable by the Sangstat Transtat®Ab kit, but not by ELISA. The remaining two patients were reactive by ELISA, but not by the Sangstat Transtat®Ab kit. This latter pattern was also observed in three healthy individuals not treated with OKT3, who exhibited high titers of anti-mouse 19 due to occupational contact with mice. The Sangstat Transtat®Ab kit required only 10-20 minutes to obtain a result whereas the ELISA required approximately 3 hours, and additional equipment. We conclude that the Sangstat Transtat®Ab kit offers the advantages of speed and ease of use for determining anti-OKT3 antibody levels in transplant recipients. However, this kit is apparently unable to detect some anti-murine antibodies.
POST-TRANSPLANT MONITORING OF HEMATOPOIETIC CELL CHIMERISM ALLOWS EARLY CLINICAL INTERVENTION FOLLOWING BONE MARROW TRANSPLANTATION. G Rosnerl, S Neudorf, E Koehl, N Zabot, M Truccol .2, IUniversity of Pittsburgh Medical Center and 2Children's Hospital of Pittsburgh, Pittsburgh, PA.
Monitoring hematopoietic cell chimerism following allogeneic bone marrow transplantation (BM1) can be helpful in deciphering the status of the patient (e.g. engrafting, relapsing) when the clinical picture is still unclear. This is especially useful in children as protocols for total body irradiation and/or T-cell depletion may result in failure to engraft or re-emergenceof the host (malignant) cells. Using a battery of 5 AMP-FLPs (Amplicon Fragment Length Polymorphisms) for minisatellite repetitive DNAs at 5 loci, we are able to find at least one informative marker for each donor/recipient pair by amplifying DNA banked in the laboratory following molecular tissue typing. DNA from posttransplant peripheral blood with WBC counts as low as 17 per p,3 can be analyzed in this assay, which can detect between 1-10% mixed chimerism. PCR products can be directly visualized on either polyacrylamide or agarose gels followed by ethidium bromide staining. Each individual shows 1 (homozygous) or 2 (heterozygous) bands per marker. This assay was used to distinguish early immunologic rejection from drug or viral-induced graft failure in several children who received T-depleted marrow transplants. This information contributed to the decision to "rescue" these patients with either a second transplant with unmodified marrow or cytokine therapy. In each case the additional therapy resulted in complete engraftment, i.e. only donor pattern was seen in this assay. The analysis of cell chimerism may also be useful in detecting early relapse. Such post-transplant monitoring allows the histocompatibility laboratory to assist the hematologist/oncologist in evaluating new protocols for BMT.
Abstracts
7.2 #186
7.2 #187
A COMPARISON OF SENSITIVITY, SPEED, AND RELIABILITY BETWEEN THE SANGSTAT TRANSTAT@AB CARTRIDGE-BASED ENZYME IMMUNOASSAY AND AN ELISA IMMUNOASSAY FOR OKT3 SERUM ANTIBODY DETERMINATIONS. JC Inlow, PK Hennessy, PW Adams, CG Orosz. The Ohio State University Hospitals, Columbus, Ohio. OKT3, an immunosuppressant used to treat acute allograft rejection, in an IgG2a mouse monoclonal antibody directed against the CD3 antigen receptor of the human T lymphocyte. Following treatment with OKT3, some patients develop antibodies against mouse Ig, which may decrease or inhibit the efficacy of monoclonal antibody treatment upon subsequent administration. Two immunoassays used to monitor anti-OKT3 antibody were evaluated: 1) a standard ELISA for human anti-OKT3 IgG, in which patient sera are added to 96 well plates coated with mouse OKT3, and serum antibody binding is detected by enzyme-conjugated rabbit anti-human IgG plus a chromogenic substrate, 2) the Sangstat Transtat®Ab kit, which is a solid phase colorimetric immunoassay in cartridge form. When tested on dilutions of pooled anti-OKT3 positive sera, both methods were equally sensitive and exhibited identical end-point titers of 1 :4000. Next, sera from 21 patients were tested with both methods, resulting in 3 discrepant results. One patient exhibited antibody (anti-idiotypic) detectable by the Sangstat Transtat®Ab kit, but not by ELISA. The remaining two patients were reactive by ELISA, but not by the Sangstat Transtat®Ab kit. This latter pattern was also observed in three healthy individuals not treated with OKT3, who exhibited high titers of anti-mouse 19 due to occupational contact with mice. The Sangstat Transtat®Ab kit required only 10-20 minutes to obtain a result whereas the ELISA required approximately 3 hours, and additional equipment. We conclude that the Sangstat Transtat®Ab kit offers the advantages of speed and ease of use for determining anti-OKT3 antibody levels in transplant recipients. However, this kit is apparently unable to detect some anti-murine antibodies.
POST-TRANSPLANT MONITORING OF HEMATOPOIETIC CELL CHIMERISM ALLOWS EARLY CLINICAL INTERVENTION FOLLOWING BONE MARROW TRANSPLANTATION. G Rosnerl, S Neudorf, E Koehl, N Zabot, M Truccol .2, IUniversity of Pittsburgh Medical Center and 2Children's Hospital of Pittsburgh, Pittsburgh, PA.
Monitoring hematopoietic cell chimerism following allogeneic bone marrow transplantation (BM1) can be helpful in deciphering the status of the patient (e.g. engrafting, relapsing) when the clinical picture is still unclear. This is especially useful in children as protocols for total body irradiation and/or T-cell depletion may result in failure to engraft or re-emergenceof the host (malignant) cells. Using a battery of 5 AMP-FLPs (Amplicon Fragment Length Polymorphisms) for minisatellite repetitive DNAs at 5 loci, we are able to find at least one informative marker for each donor/recipient pair by amplifying DNA banked in the laboratory following molecular tissue typing. DNA from posttransplant peripheral blood with WBC counts as low as 17 per p,3 can be analyzed in this assay, which can detect between 1-10% mixed chimerism. PCR products can be directly visualized on either polyacrylamide or agarose gels followed by ethidium bromide staining. Each individual shows 1 (homozygous) or 2 (heterozygous) bands per marker. This assay was used to distinguish early immunologic rejection from drug or viral-induced graft failure in several children who received T-depleted marrow transplants. This information contributed to the decision to "rescue" these patients with either a second transplant with unmodified marrow or cytokine therapy. In each case the additional therapy resulted in complete engraftment, i.e. only donor pattern was seen in this assay. The analysis of cell chimerism may also be useful in detecting early relapse. Such post-transplant monitoring allows the histocompatibility laboratory to assist the hematologist/oncologist in evaluating new protocols for BMT.