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Histopathology of biopsy in patients with anti-endothelial cell antibody and angiotensin II type 1 receptor antibody
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P038
Abstracts / Human Immunology 76 (2015) 38–167
IMMUNOMAGNETIC DEPLETION OF RBCS DIRECTLY FROM WHOLE BLOOD WITHOUT THE NEED FOR HYPOTONIC LYSIS OR SEDIMENTATION. Carrie E. Peters a, Taylor N. Lee a, Steven M. Woodside a, Terry E. Thomas a, Allen C. Eaves a,b. a STEMCELL Technologies Inc, Vancouver, BC, Canada; b BC Cancer Agency, Vancouver, BC, Canada. Aim: Sample analysis in HLA laboratories frequently requires red blood cell (RBC) depletion as a first step. However, hypotonic lysis of whole blood may be deleterious to certain cell types, and RBC removal by hetastarch sedimentation is inconsistent. Furthermore, RBC depletion with agents such as ammonium chloride is often ineffective, leaving samples that still contain many RBCs. We have developed an immunomagnetic method to deplete RBCs from whole blood samples that is rapid, effective, and leaves untouched total nucleated cells (TNC) ready for downstream assays. Methods: RBCs were removed either magnetically using the EasySep™ RBC Depletion Reagent or using ammonium chloride lysis (control). To perform magnetic RBC depletion, whole blood was diluted 1:1 with buffer (phosphate buffered saline plus 2 mM EDTA), the EasySep™ RBC Depletion Reagent added and the sample placed in an EasySep™ magnet for 5 min. Magnetically labeled RBCs were retained in the magnet and TNC were simply poured or pipetted off. The Reagent addition, magnet incubation and pour-off steps were repeated once more, followed in some cases with one more final magnet incubation and pour-off. The number of TNC recovered were counted and the samples evaluated by flow cytometry for RBC content (CD45- / Glycophorin A+) and viability (7AAD-). Results: 4:2 1:3 106 TNC per mL of starting whole blood were recovered when using the EasySep™ RBC Depletion Reagent, compared to 5:5 2:1 106 TNC recovered from the same samples by ammonium chloride lysis (n = 8; p<0.05). RBC contamination of the TNC fraction was significantly lower when samples were treated with the EasySep™ RBC Depletion Reagent rather than ammonium chloride (1.2 1.3% versus 19 18% of total events were CD45- / Glycophorin A+ respectively; p<0.05). The viability of the TNC obtained by either method was 99 1%. Conclusions: RBCs can be immunomagnetically depleted from a sample of whole blood in 10 - 15 minutes, without exposing the cells to hypotonic lysis or the need for hetastarch sedimentation. The isolated nucleated cells are immediately ready for analysis or further downstream assays. C.E. Peters: Employee; Company/Organization; STEMCELL Technologies Inc. T.N. Lee: Employee; Company/Organization; STEMCELL Technologies Inc. S.M. Woodside: Employee; Company/Organization; STEMCELL Technologies Inc. T.E. Thomas: Employee; Company/Organization; STEMCELL Technologies Inc. A.C. Eaves: Other (Identify); Company/Organization; Owner STEMCELL Technologies Inc.
P039
HISTOPATHOLOGY OF BIOPSY IN PATIENTS WITH ANTI-ENDOTHELIAL CELL ANTIBODY AND ANGIOTENSIN II TYPE 1 RECEPTOR ANTIBODY. Mary Carmelle Philogene a, Serena Bagnasco a, Denise Kielek a, Melissa Jerasano a, Inessa Kaplan a, Duska Dragun b, Edward Kraus a, Mary Sue Leffell a, Andrea A. Zachary a, Annette M. Jackson a. a The Johns Hopkins University, Baltimore, MD, United States; b Charité – Universitätsmedizin, Berlin, Germany. Aim: To compare the histologic characteristics of graft injury in the presence or absence of antibodies to endothelial cells (AECA) and angiotensin II type 1 receptor (AT1R-Ab). Method: 70 kidney recipients transplanted at the Johns Hopkins Comprehensive Transplant Center, between 1988 and 2014 were evaluated to investigate graft dysfunction. Biopsy proven rejection was defined according the Banff 2013 criteria. The specificity and level of donor specific HLA antibodies (HLA-DSA) were evaluated using HLA phenotype (Lifecodes, class I and II ID panels, Immucor, San Diego, CA) and single antigen bead panels (One Lambda, Canoga Park CA) performed on a Luminex platform. Non-HLA antibody detection was performed using quantitative ELISA (CellTrend GmbH, Luckenwalde, Germany) and precursor endothelial cell flow cytometric crossmatch, ECXM (XM-ONE Absorber AB, Stockolm, Sweden). Results: Patients were divided into three groups based on AT1R-Ab levels (positive >17 Units/ml; borderline 10–17 Units/mL and negative <10 Units/mL). AECA assessment was performed in patients whose HLA antibody was insufficient to be detected in the ECXM. There was no significant difference in patient and donor demographics among the three groups. At time of transplantation, HLA-DSA was present in 65%, 68%, and 64% of the patients in the three AT1R-Ab categories respectively. There was a greater number of positive ECXMs in the AT1R-Ab positive group compared to AT1R-Ab borderline and AT1R-Ab negative (24%, 9%, 12%). At 5 days post-transplantation, the average serum creatinine for AT1R-Ab positive, borderline and negative was 3.8, 2.3 and 1.3 respectively. At time of graft dysfunction, there was no significant difference in presence of HLA-DSA between the 3 groups (65%, 57%, 68%). The incidence of biopsy proven antibody mediated rejection was 41%, 26% and 17% for AT1R-Ab positive, borderline and negative. The average peritubular capillaritis (ptc) scores were significantly higher in the AT1R-Ab positive group compared to borderline and negative in patients who had HLA-DSA (2.3, 1.1, 0.6) and in patients with no HLA-DSA (1.7, 0.4, 0.2). Conclusion: This preliminary evaluation suggests that there is increased microvascular injury in the presence of AECA and AT1R-Ab.
P038
Abstracts / Human Immunology 76 (2015) 38–167
IMMUNOMAGNETIC DEPLETION OF RBCS DIRECTLY FROM WHOLE BLOOD WITHOUT THE NEED FOR HYPOTONIC LYSIS OR SEDIMENTATION. Carrie E. Peters a, Taylor N. Lee a, Steven M. Woodside a, Terry E. Thomas a, Allen C. Eaves a,b. a STEMCELL Technologies Inc, Vancouver, BC, Canada; b BC Cancer Agency, Vancouver, BC, Canada. Aim: Sample analysis in HLA laboratories frequently requires red blood cell (RBC) depletion as a first step. However, hypotonic lysis of whole blood may be deleterious to certain cell types, and RBC removal by hetastarch sedimentation is inconsistent. Furthermore, RBC depletion with agents such as ammonium chloride is often ineffective, leaving samples that still contain many RBCs. We have developed an immunomagnetic method to deplete RBCs from whole blood samples that is rapid, effective, and leaves untouched total nucleated cells (TNC) ready for downstream assays. Methods: RBCs were removed either magnetically using the EasySep™ RBC Depletion Reagent or using ammonium chloride lysis (control). To perform magnetic RBC depletion, whole blood was diluted 1:1 with buffer (phosphate buffered saline plus 2 mM EDTA), the EasySep™ RBC Depletion Reagent added and the sample placed in an EasySep™ magnet for 5 min. Magnetically labeled RBCs were retained in the magnet and TNC were simply poured or pipetted off. The Reagent addition, magnet incubation and pour-off steps were repeated once more, followed in some cases with one more final magnet incubation and pour-off. The number of TNC recovered were counted and the samples evaluated by flow cytometry for RBC content (CD45- / Glycophorin A+) and viability (7AAD-). Results: 4:2 1:3 106 TNC per mL of starting whole blood were recovered when using the EasySep™ RBC Depletion Reagent, compared to 5:5 2:1 106 TNC recovered from the same samples by ammonium chloride lysis (n = 8; p<0.05). RBC contamination of the TNC fraction was significantly lower when samples were treated with the EasySep™ RBC Depletion Reagent rather than ammonium chloride (1.2 1.3% versus 19 18% of total events were CD45- / Glycophorin A+ respectively; p<0.05). The viability of the TNC obtained by either method was 99 1%. Conclusions: RBCs can be immunomagnetically depleted from a sample of whole blood in 10 - 15 minutes, without exposing the cells to hypotonic lysis or the need for hetastarch sedimentation. The isolated nucleated cells are immediately ready for analysis or further downstream assays. C.E. Peters: Employee; Company/Organization; STEMCELL Technologies Inc. T.N. Lee: Employee; Company/Organization; STEMCELL Technologies Inc. S.M. Woodside: Employee; Company/Organization; STEMCELL Technologies Inc. T.E. Thomas: Employee; Company/Organization; STEMCELL Technologies Inc. A.C. Eaves: Other (Identify); Company/Organization; Owner STEMCELL Technologies Inc.
P039
HISTOPATHOLOGY OF BIOPSY IN PATIENTS WITH ANTI-ENDOTHELIAL CELL ANTIBODY AND ANGIOTENSIN II TYPE 1 RECEPTOR ANTIBODY. Mary Carmelle Philogene a, Serena Bagnasco a, Denise Kielek a, Melissa Jerasano a, Inessa Kaplan a, Duska Dragun b, Edward Kraus a, Mary Sue Leffell a, Andrea A. Zachary a, Annette M. Jackson a. a The Johns Hopkins University, Baltimore, MD, United States; b Charité – Universitätsmedizin, Berlin, Germany. Aim: To compare the histologic characteristics of graft injury in the presence or absence of antibodies to endothelial cells (AECA) and angiotensin II type 1 receptor (AT1R-Ab). Method: 70 kidney recipients transplanted at the Johns Hopkins Comprehensive Transplant Center, between 1988 and 2014 were evaluated to investigate graft dysfunction. Biopsy proven rejection was defined according the Banff 2013 criteria. The specificity and level of donor specific HLA antibodies (HLA-DSA) were evaluated using HLA phenotype (Lifecodes, class I and II ID panels, Immucor, San Diego, CA) and single antigen bead panels (One Lambda, Canoga Park CA) performed on a Luminex platform. Non-HLA antibody detection was performed using quantitative ELISA (CellTrend GmbH, Luckenwalde, Germany) and precursor endothelial cell flow cytometric crossmatch, ECXM (XM-ONE Absorber AB, Stockolm, Sweden). Results: Patients were divided into three groups based on AT1R-Ab levels (positive >17 Units/ml; borderline 10–17 Units/mL and negative <10 Units/mL). AECA assessment was performed in patients whose HLA antibody was insufficient to be detected in the ECXM. There was no significant difference in patient and donor demographics among the three groups. At time of transplantation, HLA-DSA was present in 65%, 68%, and 64% of the patients in the three AT1R-Ab categories respectively. There was a greater number of positive ECXMs in the AT1R-Ab positive group compared to AT1R-Ab borderline and AT1R-Ab negative (24%, 9%, 12%). At 5 days post-transplantation, the average serum creatinine for AT1R-Ab positive, borderline and negative was 3.8, 2.3 and 1.3 respectively. At time of graft dysfunction, there was no significant difference in presence of HLA-DSA between the 3 groups (65%, 57%, 68%). The incidence of biopsy proven antibody mediated rejection was 41%, 26% and 17% for AT1R-Ab positive, borderline and negative. The average peritubular capillaritis (ptc) scores were significantly higher in the AT1R-Ab positive group compared to borderline and negative in patients who had HLA-DSA (2.3, 1.1, 0.6) and in patients with no HLA-DSA (1.7, 0.4, 0.2). Conclusion: This preliminary evaluation suggests that there is increased microvascular injury in the presence of AECA and AT1R-Ab.