The Experts below are selected from a list of 6 Experts worldwide ranked by ideXlab platform
John L. Schmitz - One of the best experts on this subject based on the ideXlab platform.
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Rapid and quality controlled screening assay for the HLA-B57 Antigen by flow cytometry
Human Immunology, 2015Co-Authors: Tricia Crawford, Eric T. Weimer, John L. SchmitzAbstract:Aim The presence of the genotype HLA-B∗57:01 is highly predictive of Abacavir hypersensitivity. The need to identify the presence of this Antigen in the HIV population is pertinent to treatment. Traditional molecular methods of HLA-B∗57:01 with high specificity come often with long turn-around times and high costs. Our laboratory developed a flow cytometric screening method to identify the HLA-B∗57 Antigen for an expedited negative result. Methods 59 random patients were tested for the HLA-B∗57 Antigen utilizing a two-step flow cytometry staining protocol. Whole blood was incubated with CD3-APC and a biotinylated B17 monoclonal antibody. The monoclonal HLA-B∗17 antibody detects the presence of the HLA- B∗57 or B∗58 Antigens on the cell surface. To enable detection of the B57 antibody, streptavidin-PE was added. An internal positive/negative control was developed to confirm assay performance. 10 μL of Flow PRA 1 screening beads (One Lambda) were added to the patient sample. Based on SSC and FSC, beads are easily distinguishable from patient cells. All samples were confirmed by molecular HLA typing using real time PCR or SSOP. Results Of the 59 patient samples, 12 were considered positive with a Mean MFI of 8622 ± 3962. 2 of the 12 positive screens were positive for HLA-B∗57:01 by molecular typing. Based on the data, a cutoff was set with a mean MFI of 500. None of the negative screens had either HLA-B∗57 or B∗58 present by molecular typing. No cross-reactivity was observed from the 5 CREG group. Thus there is a lower likelihood of detecting Antigens outside of HLA-B∗57 and HLA-B∗58. Conclusions The screening of patients for the presence of HLA-B∗57:01 is important prior to initiation of Abacavir therapy. An estimated 0.1–2.5% of the US population is positive for HLA-B∗57:01, so the majority of patients will be negative. This rapid flow cytometry assay allows for faster turn-around time for B∗57:01 negative patients. The introduction of an internal positive control into the assay provides quality control that previous HLA-B∗57 screening assays failed to provide.
Tricia Crawford - One of the best experts on this subject based on the ideXlab platform.
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Rapid and quality controlled screening assay for the HLA-B57 Antigen by flow cytometry
Human Immunology, 2015Co-Authors: Tricia Crawford, Eric T. Weimer, John L. SchmitzAbstract:Aim The presence of the genotype HLA-B∗57:01 is highly predictive of Abacavir hypersensitivity. The need to identify the presence of this Antigen in the HIV population is pertinent to treatment. Traditional molecular methods of HLA-B∗57:01 with high specificity come often with long turn-around times and high costs. Our laboratory developed a flow cytometric screening method to identify the HLA-B∗57 Antigen for an expedited negative result. Methods 59 random patients were tested for the HLA-B∗57 Antigen utilizing a two-step flow cytometry staining protocol. Whole blood was incubated with CD3-APC and a biotinylated B17 monoclonal antibody. The monoclonal HLA-B∗17 antibody detects the presence of the HLA- B∗57 or B∗58 Antigens on the cell surface. To enable detection of the B57 antibody, streptavidin-PE was added. An internal positive/negative control was developed to confirm assay performance. 10 μL of Flow PRA 1 screening beads (One Lambda) were added to the patient sample. Based on SSC and FSC, beads are easily distinguishable from patient cells. All samples were confirmed by molecular HLA typing using real time PCR or SSOP. Results Of the 59 patient samples, 12 were considered positive with a Mean MFI of 8622 ± 3962. 2 of the 12 positive screens were positive for HLA-B∗57:01 by molecular typing. Based on the data, a cutoff was set with a mean MFI of 500. None of the negative screens had either HLA-B∗57 or B∗58 present by molecular typing. No cross-reactivity was observed from the 5 CREG group. Thus there is a lower likelihood of detecting Antigens outside of HLA-B∗57 and HLA-B∗58. Conclusions The screening of patients for the presence of HLA-B∗57:01 is important prior to initiation of Abacavir therapy. An estimated 0.1–2.5% of the US population is positive for HLA-B∗57:01, so the majority of patients will be negative. This rapid flow cytometry assay allows for faster turn-around time for B∗57:01 negative patients. The introduction of an internal positive control into the assay provides quality control that previous HLA-B∗57 screening assays failed to provide.
Yvonne M Zoet - One of the best experts on this subject based on the ideXlab platform.
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Drug induced alloreactivity: A new paradigm for allorecognition
Human Immunology, 2015Co-Authors: Lloyd D'orsogna, Paula Van Miert, Frans H.j. Claas, Mina John, Coral-ann M. Almeida, Sonia Fernandez, Yvonne M ZoetAbstract:Aim Abacavir administration is associated with drug induced hypersensitivity reactions in HIV patients expressing the HLA-B∗57:01 allele. However the immunological effects of abacavir administration in an HLA-B57 mismatched transplantation setting has not been studied. We hypothesized that abacavir exposure would induce de-novo HLA-B57 specific allorecognition. Methods Multiple HIV-specific CD8 T cell clones were generated from HIV infected patients negative for the HLA-B57 Antigen, using single cell sorting based on HIV peptide/HLA tetrameric complex staining. The generated T cell clones were assayed for alloreactivity against a panel of single HLA expressing cell lines (SALs), in the presence or absence of abacavir. Cytokine assay, CD137 upregulation and cytotoxicity were used as readout. Results Abacavir exposure did induce de-novo HLA-B57 allorecognition by HIV-specific T cells. A Gag RK9/HLA-A3 specific T cell clone, from an HLA-B57 negative HIV patient, did recognize allogeneic HLA-B57 but only in the presence of abacavir. Abacavir did not induce recognition of any other allogeneic HLA molecules. Another clone from the same patient with the same specificity, but with different TCR Vb usage, did not recognize allogeneic HLA-B57 in the presence of abacavir, suggesting TCR Vb specificity of the drug induced allorecognition. Conclusion Results presented here provide the first evidence that administration of a drug could induce specific allorecognition of mismatched HLA molecules in the transplant setting. Furthermore, HIV-specific memory T cells themselves may participate in the abacavir induced alloreactivity. We suggest that HIV-positive recipients of a HLA-B57 mismatched graft should not receive abacavir until further studies are completed.
Eric T. Weimer - One of the best experts on this subject based on the ideXlab platform.
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Rapid and quality controlled screening assay for the HLA-B57 Antigen by flow cytometry
Human Immunology, 2015Co-Authors: Tricia Crawford, Eric T. Weimer, John L. SchmitzAbstract:Aim The presence of the genotype HLA-B∗57:01 is highly predictive of Abacavir hypersensitivity. The need to identify the presence of this Antigen in the HIV population is pertinent to treatment. Traditional molecular methods of HLA-B∗57:01 with high specificity come often with long turn-around times and high costs. Our laboratory developed a flow cytometric screening method to identify the HLA-B∗57 Antigen for an expedited negative result. Methods 59 random patients were tested for the HLA-B∗57 Antigen utilizing a two-step flow cytometry staining protocol. Whole blood was incubated with CD3-APC and a biotinylated B17 monoclonal antibody. The monoclonal HLA-B∗17 antibody detects the presence of the HLA- B∗57 or B∗58 Antigens on the cell surface. To enable detection of the B57 antibody, streptavidin-PE was added. An internal positive/negative control was developed to confirm assay performance. 10 μL of Flow PRA 1 screening beads (One Lambda) were added to the patient sample. Based on SSC and FSC, beads are easily distinguishable from patient cells. All samples were confirmed by molecular HLA typing using real time PCR or SSOP. Results Of the 59 patient samples, 12 were considered positive with a Mean MFI of 8622 ± 3962. 2 of the 12 positive screens were positive for HLA-B∗57:01 by molecular typing. Based on the data, a cutoff was set with a mean MFI of 500. None of the negative screens had either HLA-B∗57 or B∗58 present by molecular typing. No cross-reactivity was observed from the 5 CREG group. Thus there is a lower likelihood of detecting Antigens outside of HLA-B∗57 and HLA-B∗58. Conclusions The screening of patients for the presence of HLA-B∗57:01 is important prior to initiation of Abacavir therapy. An estimated 0.1–2.5% of the US population is positive for HLA-B∗57:01, so the majority of patients will be negative. This rapid flow cytometry assay allows for faster turn-around time for B∗57:01 negative patients. The introduction of an internal positive control into the assay provides quality control that previous HLA-B∗57 screening assays failed to provide.
Lloyd D'orsogna - One of the best experts on this subject based on the ideXlab platform.
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Drug induced alloreactivity: A new paradigm for allorecognition
Human Immunology, 2015Co-Authors: Lloyd D'orsogna, Paula Van Miert, Frans H.j. Claas, Mina John, Coral-ann M. Almeida, Sonia Fernandez, Yvonne M ZoetAbstract:Aim Abacavir administration is associated with drug induced hypersensitivity reactions in HIV patients expressing the HLA-B∗57:01 allele. However the immunological effects of abacavir administration in an HLA-B57 mismatched transplantation setting has not been studied. We hypothesized that abacavir exposure would induce de-novo HLA-B57 specific allorecognition. Methods Multiple HIV-specific CD8 T cell clones were generated from HIV infected patients negative for the HLA-B57 Antigen, using single cell sorting based on HIV peptide/HLA tetrameric complex staining. The generated T cell clones were assayed for alloreactivity against a panel of single HLA expressing cell lines (SALs), in the presence or absence of abacavir. Cytokine assay, CD137 upregulation and cytotoxicity were used as readout. Results Abacavir exposure did induce de-novo HLA-B57 allorecognition by HIV-specific T cells. A Gag RK9/HLA-A3 specific T cell clone, from an HLA-B57 negative HIV patient, did recognize allogeneic HLA-B57 but only in the presence of abacavir. Abacavir did not induce recognition of any other allogeneic HLA molecules. Another clone from the same patient with the same specificity, but with different TCR Vb usage, did not recognize allogeneic HLA-B57 in the presence of abacavir, suggesting TCR Vb specificity of the drug induced allorecognition. Conclusion Results presented here provide the first evidence that administration of a drug could induce specific allorecognition of mismatched HLA molecules in the transplant setting. Furthermore, HIV-specific memory T cells themselves may participate in the abacavir induced alloreactivity. We suggest that HIV-positive recipients of a HLA-B57 mismatched graft should not receive abacavir until further studies are completed.