Utility of CD68/CD31 vs. CD163/CD31 Dual Immunostaining Techniques To Detect Antibody-Mediated Rejection in Post-Transplant Cardiac Biopsies.
Sergei Tatishchev, Lawrence Czer, Jon Kobashigawa, Daniel Luthringer. Cedars-Sinai MC, Los Angeles; CSMC, LA
Background: Antibody-mediated rejection (AMR) in cardiac allograft tissue predicts poor prognosis. There is no gold standard for histologic diagnosis of AMR. Identification of macrophages adherent to the vascular walls on H&E slides is indicative of AMR with ∼ 99% specificity and marginal sensitivity (30%). To offset lack of sensitivity, macrophages attached to capillaries have been visualized using antibodies against CD68 & CD31 on paraffin-embedded tissue. This approach has at least 2 intrinsic limitations: 1) Using separate tissue sections for each immunostain precludes the observer's ability to assess identical structures on both slides; 2) CD68 is an organelle-specific (i.e. lysosomes) rather than cell lineage-specific antigen, such as CD163 (macrophage). To simplify visualization of macrophages within capillaries and to assess CD68 vs. CD163 specificity we employed dual staining of paraffinized tissue with CD68/CD31 & CD163/CD31.
Design: 30 cardiac biopsies up to 3 years post-transplant were selected; 6 cases had antibody-mediated rejection (AMR1; as defined by C4d staining) and 24 were negative cases (AMR0). Sections from each case were stained with either a combination of CD68 (red chromogen) & CD31 or a combination of CD163 (red chromogen) & CD31. Dual stains performed on all AMR0 & AMR1 cases were evaluated for ability to unequivocally identify margination of macrophages within capillaries. Dual stains performed on AMR0 cases were evaluated for differences in specificity between CD68 & CD163. To assess specificity, AMR0 cases were blinded, blood vessels highlighted by CD31 on each biopsy section were counted and further stratified based on the presence or absence of distinct red chromogen-positive tissue elements within the vascular walls.
Results: Staining of AMR positive tissue sections with either CD68/CD31 or CD163/CD31 allowed a virtually effortless visualization of macrophages adherent to the capillaries in all cases. Comparison of AMR negative cases showed roughly equal specificities of 97.7% & 96.7% for CD68 & CD163, respectively.
Conclusions: Contrary to our expectations based on the reported antigenic affinities of employed immunostains, both dual staining techniques are equally sensitive and highly specific in detecting intracapillary macrophages. Dual staining using either CD163/CD31 or CD68/CD31 greatly simplifies visualization of macrophages adherent to the capillaries and is helpful for identification of AMR in post-transplant cardiac biopsies.
Wednesday, March 2, 2011 1:00 PM
Poster Session VI # 47, Wednesday Afternoon