Comparison of Flow-cytometric Antibody Secreting Cell Assay and Mabtech Immunoglobulin ELISpot Assay.

BACKGROUND: The increase of donor-specific antibodies (DSA) after transplantation could be a more important marker than the level of DSA in pre-transplantation sera. The assessment of sensitized cells that can secrete DSA is needed. We developed an assay for antibody-secreting cells (ASCs) measured with the use of flow cytometry and compared it with the Mabtech immunoglobulin (IgG) ELISpot assay. METHODS: Thirteen patients who were awaiting or received organ transplantation and 15 healthy control subjects were included. All subjects were positive for anti-cytomegalovirus (CMV) IgG. Peripheral blood mononuclear cells (PBMCs) were seeded with CpG 2006 (5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'), 500 ng/mL human CD40 ligand, and 50 ng/mL interleukin-21 in complete RPMI media. Eight micrograms of CMV pp28 antigen were added to test wells and compared with nonstimulated PBMCs. After 72 hours, analysis with the use of the human IgG ELISpot kit (Mabtech) and flow cytometry with anti-CD19-PE, CD27-PE-Cy7, CD38-APC, IgG-FITC antibodies was performed. RESULTS: The flow-cytometric ASC assay was moderately correlated with Mabtech IgG ELISpot assay (r = 0.554; P < .001). The ASCs measured by means of flow cytometry were significantly higher in healthy control subjects compared with patients (P < .001). ASCs measured by means of flow cytometry in CMV antigen-stimulated PBMCs were significantly higher compared with nonstimulated PBMCs (P < .001). The IgG-secreting cells measured by means of Mabtech ELISpot assay was not different between healthy control subjects and patients nor between CMV antigen-stimulated and nonstimulated PBMCs. CONCLUSIONS: Flow-cytometric ASC assay can differentiate ASCs for CMV antigen better than Mabtech IgG ELISpot assay. Flow-cytometric ASC assay might be useful for assessing sensitization status in patients awaiting organ transplantation.

Evaluation of Changes in New Calculated Panel Reactive Antibody Adopting HLA-Cw, DR51/52/53, and DQ Antigens in Koreans.

BACKGROUND: Calculated panel reactive antibody (cPRA) (%) is percentage of donors that would be incompatible with the candidate, based on the candidate's unacceptable HLA antigens. cPRA based on antigen frequencies of HLA-A, B, and DR has been used in Korea. We developed new cPRA including HLA-Cw, DR51/52/53, and DQ. Changes in new-cPRA were evaluated. METHODS: We analyzed the differences between cPRA based on HLA-A, -B, and -DR antigens (old-cPRA) from cPRA based on HLA-A, -B, -Cw, -DR, -DR51/52/53, and -DQ antigens (new-cPRA) on 125 waitlisted candidates for renal transplantation in Seoul National University Hospital. cPRA for unacceptable antigens was calculated according to 3 different cut-off values (MFI <1000, 3000, and 10000 for cPRAw, cPRAm, and cPRAs, respectively). RESULTS: For HLA class I, cPRAw and cPRAm were significantly increased in new-cPRA compared to old-cPRA (median 78.3% vs 71.7%, P < .001; 34.0% vs 23.5%, P = .029, respectively). For HLA class II, cPRAw, cPRAm, and cPRAs were significantly increased in new-cPRA compared to old-cPRA (median 86.8% vs 42.6%; 58.0% vs 0.0%; 0.0% vs 0.0%, P < .001 for all). CONCLUSIONS: cPRA (%) including HLA-Cw, -DR51/52/53, and -DQ showed remarkable increase, especially in HLA class II antigens. The meaning of this should be carefully interpreted through further studies considering clinical outcomes.

Anti-angiotensin type 1 receptor antibodies associated with antibody-mediated rejection in patients without preformed HLA-donor-specific antibody.

INTRODUCTION: Angiotensin II is a peptide hormone involved in the renin-angiotensin system (RAS). Anti-angiotensin receptor 1 (AT1R) antibodies are implicated in stimulating RAS and are suspected to have some adverse impacts on renal transplantation outcome. METHODS: From November 2009 to February 2012, 37 remaining sera from renal transplantation recipients with biopsy-proven antibody-mediated rejection (AMR) (n = 6), acute cellular rejection (ACR) (n = 23), and AMR + ACR (n = 8) without preformed human leukocyte antigeon (HLA) antibodies were tested with anti-AT1R antibody assay. Forty-two control patients without rejection also were analyzed. RESULTS: The frequency of elevated anti-AT1R antibodies was higher in patients with AMR (n = 14) compared to controls (28.6% vs 4.9%, P = .03, OR = 8.0). It was also higher in patients with AMR + ACR (n=8) (37.5% vs 4.9%, P = .03, OR = 12.0). There was no difference in frequencies of elevated anti-AT1R antibody in patients with ACR. CONCLUSION: Anti-AT1R antibodies were suspected to be associated with occurrence of AMR without preformed HLA antibodies in renal transplantation. Further studies in a larger number of patients are needed.