Paraffin Immunohistochemistry


Frozen tissue IF as an immunopathologic tool in evaluating AMR has been in use longer than paraffin IHC, but with modern antigen retrieval methods, IHC methods are essentially equivalent and now more commonly used in routine practice. All staining protocols must be optimized and validated appropriately and every staining run should include appropriate control tissue sections. Biopsy adequacy is also important, and the ISHLT WF recommends that 3 pieces of myocardium should be available for paraffin IHC.

Recommended vs. Optional Stains

Surveys have demonstrated wide variation in practice, highlighting the need to define appropriate antibody panel guidelines for the IHC immunopathology. The ISHLT WF proposes primary and secondary antibody panels for IHC.

Primary/Mandatory Panel


Capillary deposition of complement degradation product C4d is a key immunopathologic AMR finding. The binding of C4d to endothelial surface proteins is covalent and therefore persists much longer than other indicators of complement activation (like C1q).

Available commercial C4d antibodies for paraffin IHC are limited and almost exclusively polyclonal reagents. The following table summarizes results of a 2009 European survey of C4d staining by IHC:

There are 3 essential elements when evaluating C4d by IHC:

1. Specificity: Only interstitial capillaries in intact myocardium should be assessed. The staining of arterial, arteriolar and venular staining should be ignored, except as possible internal controls with some antibody clones. Staining of these structures should be linear (or finely granular) and encompass the entire circumference of the vessel. The terms "donut" or "elliptical" have been used when the capillaries are viewed in cross-section or tangential/longitudinal cuts, respectively.

C4d C4d


2. Intensity: Due to either assay performance or the temporal evolution of AMR the intensity of staining may vary. Occasionally, variation may be seen from region to region in the same biopsy. The 2013 ISHLT WF defines a 3-degree scale for evaluating intensity.

C4d IP 0 C4d IP 1+ C4d IP 2+ C4d IP 0 C4d IP 1+ C4d IP 2+


3. Distribution: C4d capillary staining is not always uniformly and diffusely distributed. The 2013 ISHLT WF proposes a scoring system for C4d distribution as well.

C4d IP focal C4d IP multi C4d IP diffuse

The interpretation (positive or negative) of C4d capillary depends on distribution and intensity.



Although more controversial, CD68 has been included in the primary/mandatory panel because:

  • Some cases with the same AMR histopathologic features may be C4d(-) but CD68(+).
  • Intravascular CD68+ macrophages seem to independently predict C4d deposition in capillaries and circulating DSAs.
  • In some cases, accumulating intravascular macrophages may not be apparent on routine histology, but are clearly demonstrated by CD68 staining.

    By including CD68 in the criteria for immunopathology, there is potential for "double counting" a single attribute of AMR (accumulation of intravascular monocytes). A case with this finding on routine histology that is confirmed by CD68 staining would be considered to meet both histologic and immunopathologic criteria for AMR (so pAMR2), even though complement staining may be negative.

    The essential elements in evaluating CD68 immunostaining results are:

    1. Positive cells should be restricted to the luminal spaces of capillaries and small venules. The pattern is described in the 2013 ISHLT-WF as "chain-like, linear, or beading staining profiles within interstitial capillaries or small venules aligned in longitudinal orientation and clusters or beading within the vascular lumen in cross-section". It is not always possible to distinguish intravascular from extravascular macrophages.

    CD68 IP CD68 IP CD68 IP CD68 IP CD68 IP

    2. The staining intensity of CD68+ is generally uniform, but the distribution is variable. Interpretation is further complicated by the fact that macrophages are a normal resident cell in biopsies. The 2013-WF Working Group suggests that the spectrum of "normal" includes prominent macrophages in up to 10% of the biopsy area. Beyond this, the distribution should be scored as focal (score 1): 10%-50% and multifocal/diffuse (score 2): > 50%.

    CD68 (and C4d) should be assessed on intact myocardium away from areas of well recognized ACR, Quilty lesions, biopsy site scars and areas of ischemic-related damage or necrosis. Interstitial macrophages can also be found in cellular rejection, ischemic injury, infection and healing biopsy sites. So it is important to ensure the CD68 positive cells are confined to intravascular spaces when assessing for AMR.

    There are more than one commercially available antibody clones for CD68. The KP1 clone is most commonly used in the literature, but the PGM1 clone is also acceptable. Also, CD68 has broader leukocyte specificity than some newer macrophage/monocyte lineage markers such as CD163. Studies using CD163 as an immunopathologic assessment of AMR have not yet been conducted.

    Secondary/Optional Panel


    Since C3 cleavage occurs further down the complement cascade than C4 activation, it is felt by some to be a more reliable indicator of complement cascade completion (i.e. without interruption by complement regulators). Until recently, the commercially available antibody clones for C3d could only be used in frozen section immunofluorescence. Several labs are successfully using clones in paraffin immunohistochemistry, but often report high background staining. Because experience with this is limited, C3d is not included in the primary panel for paraffin section IHC, even though it is on the primary frozen immunofluorescence panel.

    The 2013 ISHLT-WF recommends that criteria for assessing intensity and distribution of C3d staining should be the same as those for C4d evaluation. C3d findings should not be considered in the immunopathologic interpretation of AMR (since it is an optional marker).

    C3d IP


    These antibodies are very useful in assessing integrity and density of the microvascular network and provide a basis for comparison for a detailed evaluation of C4d and C3d percentage distribution, especially when digital image analysis algorithms or double staining strategies are used.

    CD31 low CD31 hi CD31 hi

    Both antibodies have well established protocols since they are widely used in surgical pathology. They are essentially interchangeable for endothelial staining although CD34 may also react with some leukocytes and other cells as well.


    This pan T-Cell antigen has been included in the secondary or optional panel since it is used by some as an attempt to better characterize the inflammatory cell component, especially the mononuclear cells within capillaries. There is morphologic overlap between some patterns of cellular rejection and the histologic features of AMR. CD3 may also be helpful in establishing the diagnosis of "mixed" rejection (concomitant acute cellular rejection and AMR). It should be noted that the ISHLT-WF experts agreed that it was not necessary to use CD3 staining routinely for the diagnosis of acute cellular rejection.


    CD20 & CD138

    Antibodies directed against the pan B-Cell (CD20) and plasma cell specific (CD138) antigens have also been included in the secondary IHC panel since they are used by some to better characterize inflammatory cell constituents. In the setting of a significant number of positive CD20 and/or CD138 cells, the possibility of lymphoproliferative disorder should be addressed (and additional studies such as EBV considered). Some pathologists also use CD20 to help differentiate Quilty effect from rejection.



    This antibody has been used as a surrogate for the detection of membrane attack complexes on capillary endothelium. It is applied for monitoring of therapeutic usage of specific humanized monoclonal antibodies (such as eculizumab). With these therapies there may be initiation of the complement cascade (so positive staining for C4d and C3d), but blockade of the cleavage of C5 step prevents it from running to completion (so no formation of membrane attack complexes). Only a few centers have experience with immunohistochemical staining of paraffin sections and since use of eculizumab and similar therapies is quite limited, this stain is considered optional.

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