Frozen Section Immunofluorescence


Frozen tissue IF has been a longstanding immunopathologic tool to evaluate AMR. The ISHLT-WF also includes recommended and optional panels for IF stains.

C4d and C3d

Other than the differing techniques and microscopy methods, the interpretation of C4d and C3d stains is identical to IHC techniques. Specifically, capillary staining should be identified being mindful of the percent of tissue involved and staining intensity.

C4d IF C3d IF C4d IF focal C4d IF multi C4d IF diffuse

Optional Stains

Other complement components (C1q/C3c)

Before C4d and C3d antibody clones were commercially available (C4d was not widely used before 1995), many laboratories were using other complement products that are commonly employed in kidney and skin biopsy immunofluorescence. Results were variable, but in general staining for C1q and C3c are considered to be too transient to be reliably detected for very long after complement activation. C1q and C3 are not felt to have a role in routine diagnosis of AMR.

A recent modification of flow cytometry based donor specific antibody (DSA) tests uses reagent complement to "spike" samples in order to determine if a detected anti-donor antibody is able to fix complement or not (by subsequently detecting C1q by flow cytometry). This in vitro procedure is very different from what occurs in vivo in terms of C1q metabolism. C1q binding is transient in vivo and staining biopsies for C1q cannot reliably provide the same data regarding complement fixation as the C1q DSA assays.

Ig Heavy Chains

Staining for immunoglobulin heavy chains (IgG, IgM, IgA) was also performed by some laboratories before complement antibodies were widely available. Staining for heavy chains may be seen in biopsies early after transplant in more severe cases and depending on the timing of the biopsy in relation to the oscillation of DSA titers, staining for heavy chains may also be seen later on. Staining for heavy chains detects antibody still bound to endothelium, a phenomenon that is also recognized to be more transient than the covalent binding of C4d. Because of this limitation, routinely staining for IgG, IgM, and IgA is not recommended in routine AMR surveillance by immunopathology.


In more severe rejection episodes the degree of endothelial damage may be so significant that coagulation cascade initiation also occurs. This rarely results in overt occlusive thrombosis of vessels, but more subtle deposition of fibrin in capillaries and leaking out of damaged capillaries into the surrounding interstitium has been described in the literature. When this pattern is seen, it indicates a more severe episode of AMR and patients whose biopsies have shown positive fibrin staining have a worse prognosis in small single center studies. The additive prognostic value of fibrin over simply recognizing higher grades of rejection in the ISHLT-WF has not been rigorously studied. This IF stain is not widely used.

fibrin fibrin



This stain is similar to CD31/CD34 by paraffin immunohistochemistry and has similar utility. The more important significance of HLA-DR staining, however, is reported to be its ability to highlight subtle damage to capillaries and has been touted as a means of assessing capillary integrity. While quiescent capillaries show crisp linear staining of endothelium, injured capillaries show more of a "frayed" or "feathered" pattern of staining with stellate projections from the capillaries into the surrounding interstitium. This pattern is thought to be helpful in identifying more severe episodes of AMR as well as "chronic AMR".

HLA-DR crisp HLA-DR feathered


Other (regulators of complement)

Activation/initiation of the complement system does not always result in its completion with membrane attach complex formation. There are several checkpoints and extrinsic regulators of the catalytic events that can prevent this. Some of these are thought to play a role in the development of immunologic tolerance. Some laboratories have developed immunofluorescence staining protocols for some of these factors such as CD59 and CD55/decay accelerating factor (DAF). The commercially available antibodies for these are limited and need further development (most require overnight incubations) before they can practically be used in routine diagnosis. The staining patterns for these are described as granular and limited to capillaries. Interpretation is more difficult than for C3d and C4d.


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