C4d is a component of classical complemet pathway which activated by immune complexes. C4d has become an essential investigation for AMR according to Banff update 2017
C4d positive in the following conditions
1. Acute AMR.
2. Chronic antibody‑mediated rejection.
3. A, B, O blood group ABO incompatible grafts.
4. Accommodation.
C4d negative in the following conditions.
1. T‑cell‑mediated rejection (TCMR)
2. A technical error.
3. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection.
4. Antibodies unable to fix the complement.
5. Complement independent pathways of endothelial activation.
6. C4d deposition is a very low in quantity for the identification limits of IF/IHC.
7. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC).
8. Increased expression of endothelial transcripts causing endothelium injury
In acute AMR, C4d is deposited in peritubular capillaries and glomerular capillaries. The diagnosis of acute AMR involves positive DSA in plasma, C4 complement component (C4d) in peritubular capillaries and structural corroboration of AMR. Transplant glomerulopathy is a variant of chronic AMR and characterized by deposition of C4d.
In conclusion, C4d‑positive staining in peritubular capillaries is a diagnostic and prognosis marker for renal allograft. The advantages for c4d include assist in diagnosis of allograft rejection, core diagnostic tool to identify AMR, and used in research into the deposition patterns of C4d in different clinical settings such as pregnancy, thrombotic complications. The disadvantages include difficulties of interpreting focal staining patterns, relatively low sensitivity of C4d as a marker for AMR in late renal allograft biopsies, and less clinical relevant as marker for antibody‑mediated injury in biopsies of ABO‑incompatible allograft.
Nandita Sugumar
2 years ago
RELEVANCE OF COMPLEMENT C4D STAINING IN RENAL ALLOGRAFT BIOPSIES
SUMMARY
AMR leads to graft failure. DSA presence is associated with early and late graft dysfunction.
C4 is part of complement pathway. It is cleaved and the active part combines with other molecules to form C4d.
Presence of C4d marks complement activation.
Clinical relevance of C4d includes the following conditions – acute AMR, chronic AMR, ABOi graft, accommodation.
The two techniques used for C4d detection include IHC and IF.
Accommodation is an important condition to consider post renal transplant. It can be defined as C4d deposition in PTC in the absence of active rejection with or without DSA positivity.
TMA is a complication of AMR.
C4d is both diagnostic and prognostic marker for allograft.
Besides rejection, C4d deposition or positivity can also be seen in pregnancy, thrombotic complications.
Chronic or late AMR can have C4d negative report.
ABOi transplants can reveal C4d negativity even in the face of allograft injury or rejection.
C4d is best interpreted along with other evidence for rejection such as biopsy features and DSA levels.
ahmed saleeh
2 years ago
Complement 4d (C4d) (that is part of component C4) is a fragment of the classical complement pathway, which is activated by immune complexes.C4 is cleaved by C1s into C4a and C4b, exposing a sulfhydryl group. The active sulfhydryl group of C4b rapidly forms an amide or ester bond with nearby molecules containing hydroxyl or amino groups and forms C4d .
C4d positivity:
a. Acute AMR b. Chronic antibody‑mediated rejection c. A, B, O blood group ABO incompatible grafts d. Accommodation.
complement 4d is negative :
a. T‑cell‑mediated rejection (TCMR)
b. A technical error like a type of fixatives, immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing endothelium injury.
Two techniques are mostly used for identification of C4d IHC and IF in allograft biopsies of the kidney.
The scoring of C4d is significant by IF on frozen section ie C4d2 or C4d3 and by IHC C4d >0 on paraffin sections.
The circulating DSA are most specifically associated with C4d deposition in PTC and its interactivity with endothelial cells in the graft.
The accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity, mostly seen in ABO‑incompatible graft transplantation.
Despite all pitfalls C4d is excellent marker for AMR.
AMAL Anan
3 years ago
Please summarise this article
Post kidney transplant, the allograft is affected by many stimulating agents such as antigen‑ antibody‑mediated immunocomplexes and cellular immunity mediated by various cells (macrophages and lymphocytes), which could lead to graft rejection.
-Complement is an important component of innate and adaptive immunity in our body. Complement is the leading effector constituent of adaptive humoral immunity.
-Following conditions are showing C4d positivity:
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation.
*The state where complement 4d is negative
The following conditions are showing C4d negativity:[10‑14]
a. T‑cell‑mediated rejection (TCMR)
b. A technical er ror li ke a type of fixatives,
immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing
endothelium injury
*The C4d‑positive staining in PTC is a diagnostic marker as well as a prognostic marker for allograft. According to various studies worldwide, C4d appears to be a less sensitive marker than initially thought.The C4d as a biomarker has following
PROS (1) provoked an enormous amount of insight in the diagnosis of allograft rejection, (2) core diagnostic tool to identify AMR, and (3) used for vast amount of research into the deposition patterns of C4d in different clinical settings
such as pregnancy, thrombotic complications and CONS as follows: (1) difficulties of interpreting focal staining patterns, (2) relatively low sensitivity of C4d as a marker
for AMR in late renal allograft biopsies, and (3) its lack of utility as a marker for antibody‑mediated injury in biopsies of ABO‑incompatible allografts). However, in which cases C4d not helpful in diagnosis whereas molecular studies have furnished perceptiveness evocative of a complement‑independent form of AMR or C4d‑negative AMR. According to us, the detection of AMR should be best reported based on morphological features such as tubulointerstitial, vascular, and glomerular histological
changes, with a piece of legislation to the presence or absence of C4d. Despite all pitfalls C4d is excellent marker for AMR.
References
1. Nankivell BJ, Alexander SI. Rejection of the kidney allograft. N Engl J Med2010;363:1451‑62.
2. Mengel M, Sis B, Haas M, Colvin RB, Halloran PF, Racusen LC, et al. Banff 2011 Meeting report: New concepts in antibody‑mediated rejection. Am J Transplant2012;12:563‑70.
3. Tait BD, Süsal C, Gebel HM, Nickerson PW, Zachary AA, Claas FH, et al. Consensus guidelines on the testing and clinical management issues associated with HLA and non‑HLA antibodies in transplantation. Transplantation 2013;95:19‑47.
4. Monsinjon T, Gasque P, Chan P, Ischenko A, Brady JJ, Fontaine MC. Regulation by complement C3a and C5a anaphylatoxins of cytokine production in human umbilical vein endothelial cells. FASEB J 2003;17:1003‑14.
5. Chakravarti DN, Campbell RD, Porter RR. The chemical structure of the C4d fragment of the human complement component C4. Mol Immunol 1987;24:1187‑97.
6. Racusen LC, Colvin RB, Solez K, Mihatsch MJ, Halloran PF, Campbell PM, et al. Antibody‑mediated rejection criteria‑An addition to the Banff 97 classification of renal allograft rejection. Am J Transplant 2003;3:708‑14.
7. Magil AB, Tinckam K. Monocytes and peritubular capillary C4d deposition in acute renal allograft rejection. Kidney Int 2003;63:1888‑93.
Nasrin Esfandiar
3 years ago
AMR due to DSA antigens is an important cause of graft loss. C4d in renal biopsy demonstrates classic complement pathway activation by Ag – Ab complexes. C1q Splits C4 to C4a and C4b to and then forms C3 convertase (C4b C2a) to split C3. There reactions finally form C5 convertase (C4b C2a C3b), that splits C5 to C5a and C5b and forms MAC. Other ways for complement activations are lectin and alternative pathways. C4d staining is positive in:
1) Acute ABMR 2) Chronic ABMR 3) ABOi 4) Accommodation:
There are 8 conditions which are C4d negative:
1) TCMR
2) Technical errors
3) FCR II A on NK cells causing rejection
4) non-complement fixing Abs
5) Very low deposition of C4d
6) Allo-Abs directly cause endothelium injury
7) Endothelia activation independent of complement pathways.
8) Expression of endothelial transcripts.
Sensitized patients are at risk of AMR. There forms of AMR are hyper-acute, acute and chronic. Acute form is related to MHC class I and II and chronic form is associated with class II. Other Ags like MICA, MICB and ABO antigens are involved in same cases.
Transplant glomerulopathy means GBM duplication or multilayering. It may be associated with peritubular capillaries multilayering. C4d staining by IHC or IF on allograft biopsies in PTC shows evidence for circulating DSA. According to Banff 2017 both C4d and transcript and are alternative to DSA.
Accommodation means C4d deposition in PTCs without active rejection or positive DSA and is seen in ABOi TX.
C4d detection by IHC is specific but its detection by IF is more sensitive. Nowadays there are pros and cons about the importance of C4d as a biomarker for graft.
Theepa Mariamutu
3 years ago
C4d is one of diagnostic criteria of antibody mediated rejection.
It is a fragment of the classical complement pathway, makes covalent bond with the tissue and remain at site of complement activation for long time.
Complement is part of adaptive humoral immunity.
It is activated by 3 pathways, the classical pathway, the lectin pathway and may be activated by CRP.
Clinical relevance of C4d
Conditions were C4d is positive:
Acute antibody mediated rejection
Chronic antibody mediated rejection
ABO blood group incompatible grafts
Accommodation
Conditions with C4d negative:
T-cell mediated rejection
Fc receptor on NK cells mediated rejection
Technical error
Antibodies unable to fix complement
Complement independent pathway of endothelial activationC4d is low in quantity for Identification limits
Increased expression of endothelial transcripts causing endothelial injury.
In acute AMR, C4d is deposited in peritubular and glomerular capillaries.
Chronic AMR is characterized by transplant glomerulopathy in renal biopsy
The effector mechanism of AMR include direct effect of antibodies to MHC, complement fixation and cellular FCRs.
Diagnosis of AMR requires detection of DSA in serum, deposition of C4d in PTC and histological evidence of AMR
Absence of C4d deposition in PTC and DSA in serum decrease the probability of AMR.
Transplant glomerulopathy:
Consists of diffuse multilayering or duplication of glomerular basement membrane which is best seen by periodic acid Schiff stain and silver stain.
may associated multilayering of peritubular capillaries:
Interstitial fibrosis and tubular atrophy exist with TG
New arterial intimal fibrosis with leukocytic infiltration in intima and absence of internal elastic lamellae favors chronic rejection with TG.
C4d in biopsies:
Immunohistochemistry and immunofixation are used in identification of C4d.
IF is applied to frozen sections, C4d stain is described as widespread, strong linear circumferential PTC staining, C4d2 or C4d3 is significant score
while IHC is applied to paraffin embedded tissue used routinely in various labs, C4d staining is crisp, linear and diffuse deposition in PTC wall, C4d>0 is significant score.
IF is more sensitive while IHC is more specific, used when frozen section is not available.
C4d is not very sensitive marker for AMR.
Interobserver variation of IHC staining is improved with the binary scoring system.
According to Banff 217, C4d is an essential investigation for AMR
C4d and validated transcripts are potential alternatives to DSA in diagnosis of AMR.
C4d negative AMR:
have higher antibody expression, poor graft outcome, usually occur after one year, may be acute or chronic and have increased intrarenal endothelial gene expression.
Accommodation is defined as C4d deposition in PTC in absence of active rejection with or without DSA seen in ABO incompatible transplant.
C4d staining is diagnostic and prognostic marker
Pros:
Used in diagnosis of AMR
Used in research in deposition patterns of C4d in different clinical settings.
Cons:
Difficulties in interpreting focal staining patterns
low sensitivity
Can’t be used as marker for AMR in ABO incompatible transplant
Last edited 3 years ago by Theepa Mariamutu
MICHAEL Farag
3 years ago
Please summarise this article
The Relevance of Complement C4d Staining in Renal Allograft Biopsies Complement 4d (C4d) is a fragment of the classical complement pathway (that is a part of component C4), which is activated by antigen‑antibody complexes. The diagnosis of AMR improves by detection of the complement fragment C4d in renal biopsy, and it has included for diagnosis of AMR in the year 2003. There is more development about C4d after inclusion in the diagnostic criteria of AMR. This review aims to evaluate pathogenesis and current relevance of C4d in AMR. Pathogenesis of complement 4d (complement system and complement 4d) There are three different pathways of complement activation and their activator The conditions where complement 4d is positive Following conditions are showing C4d positivity
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation. The state where complement 4d is negative The following conditions are showing C4d negativity:[10‑14]
a. T‑cell‑mediated rejection (TCMR)
b. A technical error like a type of fixatives, immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing endothelium injury
The C4d‑negative AMR occur due to alloantibodies to MHC molecules on endothelial cells, elicit strong responses that include proliferation and activation of intracellular signaling pathways, which leads to endothelium injury. The C4d‑negative AMR also seen with high renal endothelial transcript expression in patients with alloantibody and is an indicator of active antibody‑mediated allograft damage and poor graft outcome.
Antibody‑mediated rejection
AMR can occur in three forms, namely hyperacute, acute, and chronic rejection.
The presensitization (blood transfusion, pregnancy, and prior transplant) is a primary risk
factor for hyperacute and acute rejection.
Acute AMR is most commonly occurring due to expose of MHC Class I and II HLA antigens. Whereas, chronic AMR is largely associated with Class II DSA.
Complement 4d detection in renal allograft biopsies
Two techniques are mostly used for identification of C4d IHC and IF in allograft biopsies of the kidney.
Current status Of complement 4d
C4d is not very sensitive marker of AMR, as it originally thought. Histologically, moderate to marked microvascular inflammation (MVI) correlated with dispersing C4d positive cases in contrast to focal C4d‑positive cases.
Currently, C4d has become an essential investigation for AMR according to Banff update 2017
Conclusion The C4d‑positive staining in PTC is a diagnostic marker as well as a prognostic marker for allograft. According to various studies worldwide, C4d appears to be a less sensitive marker
than initially thought.
The C4d as a biomarker has following PROS (1) provoked an enormous amount of insight in the
diagnosis of allograft rejection, (2) core diagnostic tool to identify AMR, and (3) used for vast amount of research into the deposition patterns of C4d in different clinical settings such as pregnancy, thrombotic complications
and CONS as follows: (1) difficulties of interpreting focal staining patterns, (2) relatively low sensitivity of C4d as a marker for AMR in late renal allograft biopsies, and (3) its lack of
utility as a marker for antibody‑mediated injury in biopsies of ABO‑incompatible allografts)
Despite all pitfalls, C4d is an excellent marker for AMR.
Drtalib Salman
3 years ago
In case of mixed rejection graft loss most likely due to Ab mediated rejection rather than T .cell mediated rejection .
Drtalib Salman
3 years ago
what I learn from this paper that what previously called suspicion Ab mediated rejection(absence of DSA with histopathological picture of Ab mediated rejection ) now could be confirmed as Ab mediated rejection if biopsy confirmed positive PTC4d and intr renal endothelial gene expression .
Drtalib Salman
3 years ago
Introduction
the allograft is affected by many stimulating agents such as antigen‑ antibody‑mediated immunocomplexes and cellular immunity mediated by various cells (macrophages and lymphocytes), which could lead to graft rejection, Complement 4d (C4d) (that is part of component C4) is a fragment of the classical complement pathway, which is activated by immune complexes.
The conditions where complement 4d is positive:
a. Acute AMR.
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation.
The state where complement 4d is negative:
a. T‑cell‑mediated rejection (TCMR)
b. A technical error like a type of fixatives, immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing endothelium injury.
AMR accounts for up to 30% of all posttransplant rejection events and 20%–30% graft loss at 1 year in untreated cases.
Acute AMR is most commonly occurs due to expose of MHC Class I and II HLA antigens. Whereas, chronic AMR is largely associated with Class II DSA.
There suggested that AMR is the chief cause of graft damage, which positive IHC for C4d in PTC in the combination of high levels of DSA in the serum than cellular rejection.
Transplant glomerulopathy TG: is a morphological change that is mostly seen in humoral rejection and related to immunological etiology.TG is a variant of chronic AMR and characterized by deposition of C4d.
C4d is not very sensitive marker of AMR, as it originally thought. Histologically, moderate to marked microvascular inflammation (MVI) correlated with dispersing C4d positive cases in contrast to focal C4d‑positive cases.
C4d is significant staining in 1% or more of the PTC for formalin/IHC‑IP, or 10% or more for frozen/frequency IF.
Currently, C4d has become an essential investigation for AMR according to Banff update 2017. In this update, both C4d and validated transcripts/classifiers/molecular marker can
serve as potential alternatives and complements to DSAs in the diagnosis of ABMR.
The morphology of C4d‑positive and C4d‑negative AMR having following similar features: (1) varying degrees of glomerulitis and peritubular capillaritis, frequent TCMR, (3) both may occur early or late posttransplantation,However, C4d‑negative AMR morphologically have higher intrarenal endothelial gene expression, alloantibodies expression, poor graft outcomes, usually occur after 1‑year post transplantation, and associated acute on chronic AMR.
According Banff diagnostic criteria, C4d staining in PTCs (at least 10% PTC positive is considered as significant) by IHC on paraffin sections Banff score C4d > 0 is significant. In contrast, by IFs on frozen sections Banff scores C4d2 or C4d3 is considered significant .Thus, it is indicated that C4d detection by IHC more specific and C4d detection by IF is more sensitive.
The IHC method for C4d detection feasible in formalin‑fixed, paraffin‑embedded tissue, it is used when frozen sections facility not available, IHC also useful when small biopsy tissue or tissue not available for frozen sections.The disadvantage of C4d staining by IHC are lower sensibility, nonspecific background staining, more costly and more time consuming, and it needs external controls. IF method for C4d requires extra tissue and frozen sections facility
Jamila Elamouri
3 years ago
The Relevance of Complement C4d Staining in Renal Allograft Biopsies Post-transplant Allograft is affected by many stimuli including antibody-mediated immunocomplex and cellular immunity. Antibody-mediated rejection (AMR) is a major cause of graft loss. The clinicians and pathologists now define antibodies against HLA antigens (DSA) as a remarkable cause of early and late graft dysfunction. C4 is cleaved by C1s into C4a and C4b, exposing sulfhydryl group that rapidly forms an amide or ester bond with nearby molecules containing hydroxyl or amino groups and forms C4d. C4d makes a covalent bond with tissue and stays at the site of complement activation for a long time, in comparison to other complement pieces. Identification of C4d in renal biopsy has been introduced first under diagnostic criteria of AMR in the year 2003. Pathogenesis of complement 4d (Complement System and Complement 4d) Complement is part of innate and adaptive immunity. It is an effector element of adaptive humoral immunity. Their activation depends on three different pathways; (classical, alternative, and lectin). This results in C4d which forms a covalent bond to injured tissue for a long time, and membrane attack complex (membrane-bound C5b—9) which destroys the cells. Clinical relevance of Complement 4d: 1- Conditions associated with C4d positivity: a- Acute AMR b- Chronic AMR c- A, B, O blood group ABO incompatible graft d- Accommodation. 2- C4d negative states: a- T-cell mediated rejection (TCMR) b- A technical error like a type of fixatives, IF, versus IHC c- Fc receptor (FCR) on NK cells (FcRIIA0 mediated rejection d- Non-fixing antibodies (AB that cannot fix complement) e- Complement independent pathways of endothelial activation f- C4d deposition is very low to be identified by IF/IHC g- Alloantibodies can directly damage the endothelium by interacting with MHC antigen h- An increased expression of endothelial transcripts causing endothelial injury. AMR occurs due to alloantibodies, it is also seen with high renal endothelial transcript expression and is an indicator of active antibody-mediated allograft damage and poor graft outcome. Antibody-mediated rejection: Responsible for 20—30% of graft loss. It has 3 types; hyperacute, acute, and chronic form. Presensitization is a major risk of hyperacute and acute AMR. Acute AMR is mostly occurring due to class I and II antibodies whereas, chronic AMR is largely due to Class II. Other antigens which are responsible for AMR include MICA and ABO blood group antigen. The diagnosis of AMR depends on the collaboration of histological findings (glomerulits, peritubular capillaritis, fibrinoid necrosis of arteries, and acute tubular injury), presence of DSA and C4d deposition in peritubular capillaries (PTCs). 1- Hyperacute AMR occurs within minutes to 24 hrs after transplantation, it is mainly due to preformed DSAs in high titres. The graft appears swollen, cyanotic and flaccid, microscopically, with arteritis, interstitial oedema and extensive cortical necrosis. 2- Acute AMR, C4d is deposited in PTC and glomerular capillaries. 3- Chronic AMR is characterized by TG which is defined by glomerular basement membrane duplication or splitting and mesangial expansion of glomeruli, tubular atrophy and interstitial fibrosis, and fibrous intimal thickening in arteries. Sometimes, electron microscopy also showed peritubular capillary basement membrane multilayering.
The mechanism of AMR includes:
1- Direct effects of DSA
2- 2- complement fixation
3- Cellular FCRs
Diagnosis of acute AMR depends on detection of DSA in plasma, C4d staining in PTC, and structural graft injury in presence of graft dysfunction.
Transplant glomerulopathy: TG
It is a form of chronic AMR, caused by humoral immunity. Morphologically TG consists of diffuse multilayering or duplication of the GBM. TG does not have discrete de novo or repetitive glomerular lesion, or evidence of thrombotic microangiopathies (TMA). Double contour of GBM occurs due to subendothelial accumulation of fluff (material and mesangial cells). In TG IF and IHC show, C4d deposition along the GBM presented in a minority of cases.
Transplant Glomerulopathy also associated with multilayering of peritubular capillarie
TG features consist of cytoplasmic vacuolation, broading of subendothelial space, with the existence of electron-dense floccular material. Interstitial fibrosis and tubular atrophy. New-onset arterial intima fibrosis, with leukocytic infiltration in intima and absence of internal elastic lamella, favours chronic rejection with TG.
TG was connected with the diagnosis of AMR, in the existence of either peritubular capillaritis or glomerulitis or both as well as with C4d expulsion in PTCs.
Complement 4d detection in renal allograft biopsy:
IHC and IF are techniques used to identify C4d in allograft biopsies.
IF
IHC
Applied for the Frozen section
Applied for paraffin-embedded tissue
C4d is described as widespread, strong linear circumferential PTC staining excluding the area of scar or necrosis
C4d is crisp, linear, continuous, diffuse, and lying around in PTC wall
Its strength is slighter and changeable
Have a fine granular pattern
Scoring is significant if C4d2 or C4d3
C4d > 0
C4d staining in 10% or more
C4d staining in 1% or more of PTC
More specific
Requires extra tissue
Used if biopsy small
More sensitive
Lower sensibility
Frozen section facility
Non-specific ground staining
Costly
More time consuming
Needs external control
DSA are most specifically associated with C4d staining in the PTC. current Status of complement 4d C4d is not a sensitive marker of AMR. MVI correlated with dispersing C4d positive cases in contrast to focal C4d positive cases. Currently, C4d has become an essential investigation for AMR according to Banff update 2017. In this update, both C4d and validated transcripts/classifiers/molecular markers can serve as potential alternatives and complements to DSAs in the diagnosis of ABMR. complement 4d negative antibody‑mediatedrejection The morphology of C4d‑positive and C4d‑negative AMR has similar features:
1- glomerulitis and peritubular capillaritis.
2- frequent TCMR.
3- both may occur early or late post-transplantation.
C4d negative AMR has higher intrarenal endothelial gene expression, alloantibodies expression, and poor graft outcome usually occurs after 1-year post-transplantation and is associated with acute on chronic AMR.
Another type of AMR DSA negative AMR, in which DSA cannot be detected, moderate MVI with (g+ptc) scores ≥ 2 as per Banff 2013 with or without C4d positivity.
In ABO-incompatible graft transplantation, there is C4d deposition in PTCs in the absence of active rejection with or without DSApositivity, this process call accommodation. Conclusion:
C4d appears to be a less sensitive marker than initially thought. It is a diagnostic as well as a prognostic marker for allograft. PROS of C4d:
1- Provoked an enormous insight into the diagnosis of rejection.
2- It is a core diagnostic tool for AMR
3- C4d deposition pattern used in research in different clinical settings. CONS of C4d
1- Difficulties in interpreting focal staining pattern
2- Low sensitivity of it in late renal allograft biopsies
3- Its lack of utility as a marker for antibody-mediated injury in ABO-incompatible allografts. Despite all pitfalls, C4d is an excellent marker for AMR
the system does not take tabulation therefore there is overlap between IF and IHC characters from IF until the external control are mixed
Reem Younis
3 years ago
-The Banff criteria divided the rejection broadly into antibody‑mediated rejection (AMR), and cell‑mediated rejection. AMR is a predominant cause of allograft failure.
-Complement 4d (C4d) (that is part of component C4) is a fragment of the classical complement pathway, which is activated by immune complexes. C4 is cleaved by C1s into C4a and C4b, exposing a sulfhydryl group. The C4b forms C4d. C4d stays at the site of complement activation for a longer time, in comparison to other complement pieces.
-The diagnosis of AMR is improved by identification of the complement pieces
C4d in renal biopsy.
– Donor‑specific antibodies, are now considered by pathologists and clinicians as a significant cause of early and late graft dysfunction and failure.
– Clinical Relevance of Complement 4d In the conditions where complement 4d is positive Following conditions are showing C4d positivity:
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A ,B, O blood group ABO-incompatible grafts
d. Accommodation. The state where complement 4d is negative
The following conditions are showing C4d negativity:
a. T‑cell‑mediated rejection (TCMR)
b. A technical error like a type of fixative s , immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial
activation
f. C4d deposition is very low in quantity for the
identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact
h. Increased expression of endothelial transcripts causing endothelium injury.
-The C4d‑negative AMR occurs due to alloantibodies to MHC molecules on endothelial cells, elicit strong responses that include proliferation and activation of intracellular signaling pathways, which leads to endothelium injury.
-The C4d‑negative AMR is also seen with high renal endothelial transcript expression in patients with alloantibody and is an indicator of active antibody-mediated allograft damage and poor graft outcome.
-Antibody‑Mediated Rejection(AMR) accounts for up to 30% of all posttransplant
rejection events and 20%–30% graft loss at 1 year in untreated
cases.
-AMR can occur in three forms, namely hyperacute, acute, and chronic rejection. The presensitization (blood transfusion, pregnancy, and prior transplant) is a primary risk factor for hyperacute and acute rejection.Acute AMR is most commonly occurs due to expose of MHC Class I and II HLA antigens. Whereas, chronic AMR is largely associated with Class II DSA.
-Ther antigens that are responsible for AMR included MHC antigen (polymorphic) MICA (MHC Class I‑related chain A) and ABO blood group antigens.
-Hyperacute AMR is defined as rejection that occurs within minutes or 24 h after transplantation, mostly due to preexisting DSA present in high titers present in a patient. Grossly, the graft rapidly becomes cyanotic and flaccid. In acute AMR, C4d is deposited in PTC and glomerular capillaries. In chronic AMR, histomorphology characterized by transplant glomerulopathy (TG) in renal
biopsies.
-Transplant Glomerulopathy(TG ) is a variant of chronic AMR and is characterized by deposition of C4d. Morphologically, TG consists of diffuse multilayering or duplication of the glomerular basement membrane (GBM).
In TG double counter of GBM occurs due to the accumulation of fluff such as material
-The circulating DSA is most specifically associated with C4d deposition in PTC and its interactivity with endothelial cells in the graft.
– C4d has become an essential investigation for AMR according to Banff update 2017.
-The morphology of
C4d‑positive and C4d‑negative AMR having following similar
features:
(1) varying degrees of glomerulitis and peritubular Capillaritis.
(2) frequent TCMR.
(3) both may occur early or late posttransplantation.
-C4d‑negative AMR morphologically have higher intrarenal endothelial gene
expression, alloantibodies expression, poor graft outcomes, usually occur after 1‑year posttransplantation, and associated acute on chronic AMR.
-The accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity, mostly seen in ABO‑incompatible graft transplantation.
-C4d detection by IHC is more specific and C4d detection by IF is more sensitive. The IHC method for C4d detection is feasible in formalin‑fixed, paraffin‑embedded tissue, it is used when frozen sections facility is not available, IHC also useful when
small biopsy tissue or tissue not available for frozen sections.
-The disadvantage of C4d staining by IHC are lower sensibility, nonspecific background staining, more costly and more time consuming, and it needs external controls.
– IF method for C4d requires extra tissue and frozen sections facility.
Zahid Nabi
3 years ago
The knowledge about role of C4 d in diagnosing AMR has evolved over the years . This article again has explored the relevance of C4d in this regard.
Complement 4d (C4d) (that is part of component C4) is a fragment of the classical complement pathway, which is activated by immune complexes. Complement is an important component of innate and adaptive immunity in our body. Complement is the leading effector constituent of adaptive humoral immunity.
The conditions where complement 4d is positive
Following conditions are showing C4d positivity
a. Acute AMR
b. Chronic antibody‐mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation.
The state where complement 4d is negative
The following conditions are showing C4d negativity
a. T‐cell‐mediated rejection (TCMR)
b. A technical error like a type of fixatives,
immunofluorescence (IF) versus immunohistochemistry
(IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated
rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial
activation
f. C4d deposition is a very low in quantity for the
identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact
with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing
endothelium injury.
Different techniques for detecting C4d are IF and IHC.
IF technique applied to frozen sections whereas IHC technique used to paraffin‐embedded tissue used routinely in various laboratories. In acute and chronic AMR‐positive C4d stain with IF technique is described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to a consensus at 2003 Banff Conference.[6] In IHC, C4d staining is crisp, linear, continuous, diffuse, and lying around in the PTC wall, while the strength typically is slighter and changeable, but it may have a finely granular pattern in high power.[40] The scoring of C4d is significant by IF on frozen section ie C4d2 or C4d3 and by IHC C4d >0 on paraffin sections.
According to various studies worldwide, C4d appears to be a less sensitive marker than initially thought however it should always be interpreted in a given clinical scenario
Heba Wagdy
3 years ago
Introduction
C4d is one of diagnostic criteria of antibody mediated rejection.
It is a fragment of the classical complement pathway, makes covalent bond with the tissue and remain at site of complement activation for long time.
Complement is part of adaptive humoral immunity.
It is activated by 3 pathways, the classical pathway, the lectin pathway and may be activated by CRP. Clinical relevance of C4d Conditions were C4d is positive:
Acute antibody mediated rejection
Chronic antibody mediated rejection
ABO blood group incompatible grafts
Accommodation Conditions with C4d negative:
T-cell mediated rejection
Fc receptor on NK cells mediated rejection
Technical error
Antibodies unable to fix complement
Complement independent pathway of endothelial activationC4d is low in quantity for Identification limits
Increased expression of endothelial transcripts causing endothelial injury. Antibody mediated rejection (AMR):
In acute AMR, C4d is deposited in peritubular and glomerular capillaries.
Chronic AMR is characterized by transplant glomerulopathy in renal biopsy
The effector mechanism of AMR include direct effect of antibodies to MHC, complement fixation and cellular FCRs.
Diagnosis of AMR requires detection of DSA in serum, deposition of C4d in PTC and histological evidence of AMR
Absence of C4d deposition in PTC and DSA in serum decrease the probability of AMR. Transplant glomerulopathy (TG):
Variant of chronic AMR, characterized by deposition of C4d.
Consists of diffuse multilayering or duplication of glomerular basement membrane which is best seen by periodic acid Schiff stain and silver stain. TG also associated multilayering of peritubular capillaries:
Interstitial fibrosis and tubular atrophy exist with TG
New arterial intimal fibrosis with leukocytic infiltration in intima and absence of internal elastic lamellae favors chronic rejection with TG. C4d detection in allograft biopsies:
Immunohistochemistry (IHC) and immunofixation (IF) are used in identification of C4d.
IF is applied to frozen sections, C4d stain is described as widespread, strong linear circumferential PTC staining, C4d2 or C4d3 is significant score
while IHC is applied to paraffin embedded tissue used routinely in various labs, C4d staining is crisp, linear and diffuse deposition in PTC wall, C4d>0 is significant score.
IF is more sensitive while IHC is more specific, used when frozen section is not available. Current status of C4d:
C4d is not very sensitive marker for AMR.
Interobserver variation of IHC staining is improved with the binary scoring system.
According to Banff 217, C4d is an essential investigation for AMR
C4d and validated transcripts are potential alternatives to DSA in diagnosis of AMR. C4d negative AMR:
It was included in Banff 2013 classification, Both C4d positive and C4d negative AMR have same different degrees of glomerulitis and peritubular capillaritis, frequent T cell mediated rejection and may occur early or late post transplant.
C4d negative AMR have higher antibody expression, poor graft outcome, usually occur after one year, may be acute or chronic and have increased intrarenal endothelial gene expression.
Accommodation is defined as C4d deposition in PTC in absence of active rejection with or without DSA seen in ABO incompatible transplant.
C4d staining is diagnostic and prognostic marker Pros:
Used in diagnosis of AMR
Used in research in deposition patterns of C4d in different clinical settings. Cons:
Difficulties in interpreting focal staining patterns
low sensitivity
Can’t be used as marker for AMR in ABO incompatible transplant
Introduction:
C4d is a degradation product of the classical complement pathway, which is activated by antigen antibody complexes. These alloantigens can be HLA Class 1&11, Non HLA and ABO antigens located at vascular endothelium.
C4D in the allograft biopsy facilitating diagnosis of ABMRT
Pathogenesis of C4d:
C4 is cleaved by C1s into C4a and C4b, exposing a sulfhydryl group. The C4b forms C4d which stays at the site of complement activation for a longer time, in comparison to other complement pieces.
Conditions associated with C4D positive .:
Acute ABMR.
Chronic ABMR.
ABO blood group incompatible grafts and Accommodation.
C4d Negative Causes:
T-cell Mediated Rejection (TCMR)
Technical error.
Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
Antibodies are unable to fix complement.
Complement independent pathways of endothelial activation.
C4d amount is very low which can’t be detected .
Increased expression of endothelial transcripts causing endothelium injury
Antibody Mediated Rejection (AMR):
The AMR is very harmful to the graft especially in more sensitized patients.
It accounts for up to 30% of all posttransplant rejection events and 20-30% graft loss at 1 year in untreated cases.
AMR can occur as hyperacute, acute, and chronic rejection.
Complement 4d Detection in Renal Allograft Biopsies
C4D is detected by two techniques: IHC and IF in allograft biopsies.
IF technique for frozen sections and the IHC technique used to paraffin‑sections
C4D is positive when it is C4d2 or C4d3 by IF or C4d >0 by IHC
IHC is more specific and C4d detection but IF is more sensitive.
As per Banff updates AMR is diagnosed by
morphological features such as tubulointerstitial, vascular, and glomerular histological changes
evidence of antigen antibodies interactions like C4D deposition
serological evidence of DSA
Shereen Yousef
3 years ago
•AMR is one of the major causes of graft loss
It occurs due to allo-antibodies that interact with donor antigen initiating immune responses.
It may acure as hyperacute within minutes to 24 hours, acute or chronic based on the degree of sensitization and Mismatchs between donor and recipient.
•Acute AMR most commonly occurs due to exposure to MHC Class I and II HLA antigens. chronic AMR is associated with Class II DSA.
There are also other antigens responsible for AMR includes MICA (MHC Class I‑related chain A) and ABO blood group antigens.
Complement system activationcan occure by three ways ,and is considered as the major factor of allograft injury in AMR.
C4d is a degradation product of the classical complement pathway, which is activated by antigen antibody complexes.
●conditions where complement 4d is positive
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO incompatible grafts d. Accommodation.
According Banff diagnostic criteria, C4d staining in PTCs (at least 10% PTC positive is considered as significant) by IHC on paraffin sections, Banff score C4d > 0 is significant.
by IFs on frozen sections Banff scores C4d2 or C4d3 is considered significant.
Thus, it is indicated that C4d detection by IHC more specific and C4d detection by IF is more sensitive.
●The state where complement 4d is negative: a. T‑cell‑mediated rejection (TCMR)
b. technical error .
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can directly cause endothelium injury
h. Increased expression of endothelial transcripts causing endothelium injury.
*Mixed cellular and humoral rejection are very hard to examine
AMR was considered the main cause of graft damage if there is positive IHC for C4d in PTC + high levels of DSA in the serum than cellular rejection.
*diagnostic features of AMR:
-presence of DSA andC4d deposition in peritubular capillaries (PTCs) in biopsy
– with other Microscopic findings of AMR; glomerulitis, peritubular capillaritis, fibrinoid necrosis of arteries, and acute tubular injury.
*chronic AMR, histomorphology characterized by transplant glomerulopathy (TG) in renal biopsies.
TG is a variant of chronic AMR and characterized by deposition of C4d. It proposed that TG may be one manifestation of humoral rejection of graft injury.
*The TG appears as:
capillary basement membrane duplication and mesangial expansion of glomeruli,
tubular atrophy and interstitial fibrosis,
fibrous intimal thickening in arteries. Sometimes, electron microscopy also showed peritubular capillary basement membrane multilayering
*The effector mechanisms of AMR includes
(1) Direct effects of antibody to MHC.
(2) Complement fixation.
(3) Cellular FCRs
•Current Status of complement 4d
C4d is not very sensitive marker of AMR, as it originally thought. Histologically, moderate to marked microvascular inflammation (MVI) correlated with dispersing C4d positive cases in contrast to focal C4d‑positive cases.
C4d has become an essential investigation for AMR according to Banff update 2017.
In this update, both C4d and validated transcripts/classifiers/molecular marker can
serve as potential alternatives and complements to DSAs in the diagnosis of ABMR.
•C4d negative antibody‑mediated rejection:
The C4d‑negative AMR was included in Banff 2013 classification.
The C4d‑negative AMR occur due to alloantibodies to MHC molecules on endothelial cells, elicit strong responses that include proliferation and activation of intracellular signaling pathways, which leads to endothelium injury.
The C4d‑negative AMR also seen with high renal endothelial transcript expression in patients with alloantibody and is an indicator of active antibody‑mediated allograft damage and poor graft outcome.
The morphology of C4d‑positive and C4d‑negative AMR having following similar features: (1) varying degrees of glomerulitis and peritubular capillaritis,
(2) frequent TCMR.
(3) both may occur early or late posttransplantation.
C4d‑negative AMR morphologically have higher intrarenal endothelial gene expression, alloantibodies expression, poor graft outcomes, usually occur after 1‑year posttransplantation, and associated acute on chronic AMR.
•conclusions
*The C4d‑positive staining in PTC is a diagnostic marker as well as a prognostic marker for allograft.
*C4d is less sensitive marker , some cons are :1)difficulties of interpreting focal staining patterns
(2) relatively low sensitivity of C4d as a marker for AMR in late renal allograft biopsies.
(3) its lack of utility in biopsies of ABO‑incompatible allografts.
Therefore, the detection of AMR should be best reported based on morphological features such as tubulointerstitial, vascular, and glomerular histological changes, with a piece of legislation to the presence or absence of C4d
Ramy Elshahat
3 years ago
the Banff criteria divided the rejection into antibody‑mediated rejection (AMR), and cell‑mediated rejection.
AMR is a predominant cause of allograft failure caused by antibodies against human leukocyte antigen (HLA) antigens defined as donor‑specific antibodies(DSA) which can cause early and late graft dysfunction.
(C4d) is part of component C4 is a part of the complement pathway. C4 is cleaved by C1s into C4a and C4b, exposing a sulfhydryl group which rapidly makes covalent bond with the tissue and stays at the site of complement activation for a longer time and can be used as a fingerprint for ABMR.
Pathogenesis
Complement 4d (Complement System and Complement 4d) Complement is an important component of innate and adaptive immunity in our body. Complement is the leading effector constituent of adaptive humoral immunity. There are three different pathways of complement activationand their activator.
· In the classical pathway of complement kick over of C1 (which is composed of C1q, C1r, and C1s) is begun by
1. an interplay of C1q with immunoglobulin bound to epitopes on the graft endothelium.
2. C1s(s>split) splits C4 into C4a and C4b and reacts by exposing a sulfhydryl group. The ester or amide bond forms with the interaction between the sulfhydryl group of C4b and nearby molecules containing hydroxyl or amino groups in the tissue.
3. C3 convertase (C4bC2a) of classical pathway is formed by a combination C4b with enzymatically loose pieces of C2a. C4bC2a (C3 convertase) splits C3 into C3a and C3b. C3b has an active sulfhydryl group that is dative bound and settles in the immediate locality and forms C5 convertase (C4bC2aC3b).
4. C5 convertase split C5 into C5a and C5b. Further, membrane attack complex (membrane‑bound C5b‑9) formed by the addition of C5b component of C5 convertase, that destroys cells.
· The lectin pathway of complement activatesby mannan‑binding lectin (MBL) and H‑ficolin or L‑ficolin.
1. The MBL attaches to the suitable carbohydrate on apoptotic cells or pathogens)>not Ag X Ab.
2. MBL, H‑ficolin, and is L‑ficolin are homologous to C1q and fibrinogen(C1Q not involved in MLP), which are also activated C4 by their associated serine proteases, mannose-associated serine protease 1 (MASP-1 and MASP-2) (analogs to C1r and C1s).
· The C‑reactive protein (CRP) also activates the complement (C4)pathway. The CRP binds to carbohydrate and choline phosphate along with C1q (INVOLVED).
The following conditions are showing C4d positivity:
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO-incompatible grafts
d. Accommodation.
The following conditions are showing C4d negativity:
1. T‑cell‑mediated rejection (TCMR)
2. A technical error like a type of fixatives, immunofluorescence (IF) versus immunohistochemistry (IHC)
3. C4d deposition is a very low in quantity for the identification limits of IF/IHC
4. complement independent mechanism include Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection and endothelial activation as alloantibodies can direct endothelium injury to interact with major histocompatibility (MHC) Increased expression of endothelial transcripts causing endothelium injury.
5- Antibodies unable to fix the complement (IgG 2,4) complex
The C4d‑negative AMR is also seen with high renal endothelial transcript expression in patients with alloantibody and is an indicator of active antibody-mediated allograft damage and poor graft outcome.
Antibody‑Mediated Rejection
The AMR harms allograft kidney, chiefly in more sensitized patients. AMR accounts for up to 30% of all posttransplant rejection events and 20%–30%graft loss at 1 year in untreated cases.
AMR can occur in three forms, namely hyperacute, acute, and chronic rejection. The presensitization (blood transfusion, pregnancy, and prior transplant) is a primary risk factor for hyperacute and acute rejection.
Complement 4d Detection in Renal Allograft Biopsies
Two techniques that are mostly used in the detection of C4d include IHC and IF in allograft biopsies. IF technique is applied to frozen sections whereas the IHC technique used to paraffin‑sections
IF technique there is linear deposition along with PTC”
In IHC, C4d staining is linear, and diffuses along the PTC wall but it may have a finely granular pattern in high power.
The scoring of C4d is significant by IF on frozen sections ie C4d2 or C4d3 and by IHC C4d >0 on paraffin sections.
IHC is more specific and C4d detection by IF is more sensitive.
The disadvantage of C4d staining by IHC are lower sensibility, nonspecific background staining, more costly and more time consuming, and it needs external controls.
The C4d as a biomarker has the following PROS:
(1) specific in the diagnosis of ABMR
(2) deposition patterns of C4d and sites of deposition can be used in DD CONS :
(1) focal staining patterns make it difficult in interpretation
(2) low sensitivity
(3) not useful in biopsies of ABO‑incompatible allografts.
as per Banff updates
AMR diagnosis based on the presence of all 3 criteria
morphological features such as tubulointerstitial, vascular, and glomerular histological changes
evidence of antigen antibodies interactions like C4D deposition
serological evidence of DSA and C4D
so Despite all pitfalls, C4d is an excellent marker for AMR
Mahmud Islam
3 years ago
aftre renal transplantation, in case of the presence of antibodies, immune complexes form and react with real endothelium resulting in rejection and graft loss. this is hyperacute if happens within minutes up to 24 hours. other scenarios are acute and chronic rejection. hyperacute is usually due to preformed antibodies. acute AMBR can be due to either class I or II while chronic rejection is usually due to class II.
c4d is a by-product of the classical pathway. c4d is usually stained positive in case of acute AMBR, chronic ABMR, ABO-incompatible grafts, and in accommodation (mostly seen in ABO-incompatible transplants). accommodation is defined as C4d deposition in PTC in the absence of active rejection with or without DSA. C4d may be negative despite rejection. this may be cellular rejectşon or antibody-mediated but due to a third party. some antibodies are non-complement fixing. low levels of complement (below the threshold of detection). alloantibodies may lead to c4d -ve ABMR.
In acute AMBR c4d is deposited in PTC (peritubular capillaries) and glomerular capillaries. chronic ABMR is characterized by (TG) transplant glomerulopathy. TG is chracterşsed by duplication and multilayering of BM.
later both mixed cellular and ABMR may coexist but dşfferentiaition is hard because of the coexistence of both acute and chronic levels. ABMR is usually discarded in case of undetected DSAs and c4d negativity.
TG is a variant of chronic ABMR.
complement 4d is detected by either IF or IHC. IF is detected in frozen sections while IHC is used for paraffin-embedded tissue.
c4d negative ABMR included first in Banff 2013 has the same morphology as positive ABMR (varying degrees of glomerulitis and peritubular capillaritis, frequent TCMR and may occur early or late. however, c4d negative ABMR may have poor outcomes.
Mohamed Essmat
3 years ago
-Summary: Banff criteria classify rejection into AMR and TCMR. In AMR with +ve DSAs immune complexes activate the classical complement pathway giving rise to C4d. Rejection with –ve C4d negative stain in the presence of AMR can be due to technical error , Fc receptor on NK cell mediated rejection, non-complement fixing antibodies, low quantity of C4d deposits ,non-HLA antibodies, and in antibodies causing direct endothelial injury. Past history of sensitization is associated with hyper-acute and acute rejection. Acute AMR is associated with both class I and II DSA while chronic AMR is mainly associated with class II DSA. Histopathology of AMR include glomerulits, peritubular capillaritis, fibrinoid necrosis and acute tubular injury and C4d deposition in peritubular capillaries. C4d deposition is less in TG. It has been shown that C4d is not a very sensitive marker for AMR. In Banff 2017 update, for a diagnosis of AMR, in addition to DSA and biopsy findings , either C4d or molecular markers can be used.
Nandita Sugumar
3 years ago
RELEVANCE OF C4d STAINING IN RENAL ALLOGRAFT BIOPSY SUMMARY
The given article looks into the formation of C4d and its role in pathogenesis of AMR and its identification via biopsy.
C4d is a part of the complement pathway C4 and is formed by cleavage of C4 into the C4d and C4d to form C4d. C4d covalently bonds with the graft tissue and AMR can be diagnosed through testing for C4d presence via renal graft biopsy.
C4d positive is seen in following conditions
Acute AMR
Chronic antibody mediated rejection
A,B,O blood group ABO incompatible grafts
accommodation
C4d negative AMR is seen in following conditions
TCMR
Technical error
FCR on NK cells mediated rejection
Endothelial activation independent of complement pathways
Low C4d deposition
Endothelial injury due to increased expression of tissue transcripts
AMR affects the allograft mainly in previously sensitized patients. It can occur as hyper acute, acute or chronic rejection. Hyperacute and acute rejection is seen in presensitized patients.
Transplant glomerulopathy is characterized by C4d deposition and is a variant of chronic AMR. Diffuse multilayering or duplication of the glomerular basement membrane is a distinct feature of this type of rejection. This duplication can be recognized with periodic acid Schiff staining methods.
C4d is a good tool in diagnosing AMR, both acute and chronic. However, it is not the best marker available and is less sensitive than it is thought to be.Discerning differences in focal patterns, low sensitivity in late biopsies as well as lack of diagnostic power in terms of ABO incompatible transplants all serve as points against C4d. However it is overall an excellent marker for AMR which should be included in making the ultimate diagnosis of rejection.
Murad Hemadneh
3 years ago
The Relevance of Complement C4d Staining in Renal Allograft Biopsies. Introduction:
C4d is a degradation product of the classical complement pathway, which is activated by antigen antibody complexes. Complement system activation is the major contributor of allograft injury in antibody mediated rejection which can occur by three major pathways: classical, lectin, and alternative. These alloantigens can be HLA Class 1&11, Non HLA and ABO antigens located at vascular endothelium. The diagnosis ABMR is facilitated by incorporation C4d in the allograft biopsy. This review aims to evaluate pathogenesis and current relevance of C4d in ABMR.
Pathogenesis of C4d: Classical pathway is activated when the C1 complex (C1q, C1r and C1s serine proteasess), binds to the Fc region of complement-fixing antibodies attached to the antigen. Activation of C1r and C1s in turn cleaves C4 and C2 into larger (C4b, C2a) and smaller (C4a, C2b) fragments.. C4b is converted to C4d which is main hallmark of ABMR. C4d staining has an important role in diagnosis of Acute and chronic ABMR.
C4d positive associated condition:
Acute ABMR.
Chronic ABMR.
ABO blood group incompatible grafts and Accommodation.
C4d Negative Causes:
T-cell Mediated Rejection (TCMR)
Technical error.
Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
Antibodies are unable to fix complement.
Complement independent pathways of endothelial activation.
C4d amount is below the threshold for detection.
.Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC).
Increased expression of endothelial transcripts causing endothelium injury
Antibody Mediated Reaction (AMR):
AMR can occur in three forms: hyperacute, acute, and chronic rejection.
AMR accounts for 30% of all transplanted patients, and graft loss at 1 year occurred in 20-30% of the cases if remained untreated.
Acute AMR occurs due to both class & class II antibodies. Graft is swollen due to interstitial oedema & haemorrhages. Histologic findings are glomerulitis, peritubular capillaritis, ATI and intimal arteritis. Associated with positive serum DSA and positive staining of C4d in biopsy.
Chronic AMR mostly due to class II. It’s associated with circulating DSA and positive C4d staining in Peritubular capillaries. Histological findings are transplant glomerulopathy, multilayering of peritubular cell basement membrane, chronic arteriopathy and absence of acute inflammation.
Patients with DSA who are C4d positive have lower graft survival than those who are C4d negative, this means that it is a marker for severity and has prognostic implication.
C4d negative patients have a higher rate of developing transplant glomerulopathy if untreated.
Transplant Glomerulopathy (TG):
TG is an irreversible histologic finding that occur in late stages of ABMR.
Present by gradual decline of renal functions together with proteinuria.
Once occur it indicate poor graft survival.
TG is a variant of chronic AMR and characterized by deposition of C4d, new basement membrane formation in absence of thrombotic microangiopathies (TMA), peritubular capillaries (PTC) multi-layering and arterial intimal fibrosis.
C4d positivity can be variable and diagnosed at later stages of AMBR.
Conclusion
C4d is still an indicative but less sensitive marker for allograft rejections as though previously.
High expression of endothelial transcript on biopsy may be useful in cases of negative C4d staining.
Esmat MD
3 years ago
Anti-HLA DSAs are essential in both early and late graft survival. C4d is a fragment of complement classic pathway. It remains at the site of complement activation for a long time. Diagnosis of C4d in kidney biopsy has provided better yield for diagnosis of AMR. The complement system is a crucial component of innate and adaptive immunity. The complement system has three different path way.
Activation of the classical pathway starts with the interplay of C1q with immunoglobulin bound to epitopes on the graft endothelium. C4 is split into C4a and C4b and then C4b is converted to C4d. Activation and continuation of complement cascade by formation of C3 convertase and C5 convertase ultimately leads to formation of MAC and cell lysis. The lectin pathway, which is activated by MBL, H-ficolin, and L-ficolin also activates C4. In addition, CRP activates the complement C4 pathway.
The conditions with positive C4d staining are acute AMR, chronic AMR, ABO incompatible transplantation (accommodation).
The conditions with negative C4d staining include TCMR, technical errors, FCRII mediated rejection, non-complement fixing antibodies, very low quantity of C4d deposition, direct endothelium injury by alloantibodies, increased expression of endothelial transcripts.
C4d negative AMR due to antibody against MHC molecules on endothelium that are correlated with high endothelial transcripts expression can lead to severe endothelium injury and poor graft outcomes.
AMR
AMR constitutes up to 30% of posttransplant rejection. There are three forms of AMR. Acute AMR is commonly associated with class I and class II HLA antigens and chronic AMR is mostly associated with class II HLA antigens. MICA and ABO antigens are also responsible for AMR. The peritubular C4d finding is one of pathological diagnostic features of AMR (besides peritubular capillaritis, glomerulitis, fibrinoid necrosis, and acute tubular injury). Hyper acute rejection that occurs during the first 24 hours after transplantation is characterized by arteritis, interstitial edema, and extensive cortical necrosis. In chronic AMR, transplant glomerulopathy with duplication or splitting of GBM, mesangial expansion and IFTA are characteristic features. C4d deposition may be seen along with the GBM and peritubular capillary multilayering in EM may also be seen in TG. Mixed cellular and humoral rejection is hard for diagnosis, and positive peritubular C4d staining in combination of high level of DSA is highly suggestive of AMR. Excessive NK cells have been seen in the biopsy of acute and chronic AMR. The frozen section IF technique and paraffin-embedded IHC technique are used for detection of C4d. C4d staining by IF described as widespread, linear circumferential PTC in cortex and medulla, sparing scar and necrosis, and by IHC described as script, linear, continuous PTC and finely granular in high power.
C4d is not a very sensitive marker of AMR. Moderate to marked MVI is associated with disseminated C4d positivity. in Banff 2017, C4d, transcripts and molecular markers are considered as an alternative or complement of DSA.
C4d negative AMR
Although C4d positive and C4d negative have some similar features such as glomerulitis and peritubular capillaritis, C4d negative AMR have higher intrarenal endothelial gene expression and is usually associated with late acute AMR and chronic AMR.
AMR DSA negative AMR is described in the absence of DSA and presence of moderate MVI. TMA is a complication of AMR. C4d staining in peritubular capillaries more than 10% by IF and more than 0 by IHC is considered significant. C4d detection by IHC is more specific and by IF is more sensitive. IHC is also useful for small tissue biopsy, but is related to higher cost, nonspecific background staining, and higher time consuming.
Dalia Eltahir
3 years ago
(C4d) is a fragment of the classical complement pathway, which is activated by immune complexes.
identification C4d in renal biopsy improved the diagnosis of AMR . C4d positive in flowing condition
a. Acute AMR
b. Chronic antibodymediated rejection
c. ABO incompatible grafts
d. Accommodation.
C4d negative in flowing condition
a. TCMR
b. Technical error
c.minimal C4d deposition
d. Antibodies cannot be attached to complement
e. Complement independent pathways of endothelial
activation
f.injury by Alloantibodies AntibodyMediated Rejection: accounts for about 30% of all post-transplant rejection events and 20%–30% graft loss at 1 year if untreated.There are three forms of AMR: hyperacute, acute, & chronic . Acute AMR associated with HLA Class I & II antigens while chronic AMR is associated with Class II DSA.
Diagnosis of acute AMR : Diagnosis by DSA level ,C4d deposition in PTC, clinically rising scr . Transplant Glomerulopathy: duplication of the glomerular basement membrane (GBM) and interstitial fibrosis and atrophy.
Two techniques used for identification of C4d. C4d detection by IHC is more specific & C4d detection by IF is more sensitive . PROS :
(a) provoked an enormous amount of insight in the diagnosis of allograft rejection;
(b)It is core diagnostic tool to identify AMR;
c) used for majority of researches into the deposition patterns of C4d in different clinical settings such as pregnancy, thrombotic complications CONS:
a) difficulties of explanation focal staining patterns;
(b) low sensitivity of C4d as a marker for AMR in late renal allograft biopsies;
(c) its lack of utility as a marker for antibodymediated injury in biopsies of ABOincompatible allografts)
Abdullah Raoof
3 years ago
AMR IS apredominant cause of allograft loss. C4d is a component of C4 complement factor . its presence suggest activation of classical complement pathway . detection of C4d in biopsy improves AMR diagnosis .thas wy it is included in diagnosis of AMR in Banff 2003.
C4d makes covalent bond with tissue and stay in tissue for longer time .
Following conditions are showing C4d positivity:
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation.
The following conditions are showing C4d negativity:
a. T‑cell‑mediated rejection (TCMR)
b. A t e ch n ica l e r ror l i ke a t y p e of f i x at ive s , immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing endothelium injury.
AMR ACOUNT FOR 30% acute rejection .and 30% of graft loss at 1 year.
Acute AMR is associated with both class I and class II HLA Ag. Whereas chronic rejection largely associated with class I .
Hyperacute AMR is defined as rejection occurs within minutes
or 24 h after transplantation, mostly due to preexisting DSA Acute AMR, C4d is deposited in PTC and glomerular capillaries. In Chronic AMR, histomorphology characterized by transplant glomerulopathy (TG) in renal biopsies.
The effector mechanisms of AMR
(1) Direct effects of antibody to MHC,
(2) Complement fixation,
(3) Cellular FCRs
Diagnosis of AMR based on three component
1- Presence of DSA
2- Feature of Ab –Ag reaction ( c4d …)
3- Hitopathological signs of rejection.
Some studies had shown by utilizing microarray investigation showed excessive NK cells in biopsies of patients with acute and chronic AMR .
Transplant Glomerulopathy
TG is a morphological change that is mostly seen in humoral rejection and related to immunological etiology. TG is a variant of chronic AMR and characterized by deposition of C4d. TG consists of diffuse multilayering or duplication of the glomerular basement membrane .
Glomerular duplication is best seen by silver and PAS stain .
TG was connected with the diagnosis of AMR, in the existence of either peritubular capillaritis or glomerulitis or both as well as with C4d expulsion in PTCs
C4d is detected by two technique
IF – is asensitive but not specific test ,it needs fresh frozen sample , and adequate tissue sample .
IHC – is more specific than IF but less sensitive , more costy, more time consuming ,can be done in paraffin embedded tissue .
The scoring of C4d is significant by IF on frozen section ie C4d2 or C4d3 and by IHC C4d >0 on paraffin sections.
Currently, C4d has become an essential investigation for AMR
according to Banff update 2017.
The morphology of C4d‑positive and C4d‑negative AMR having following similar features:
(1) varying degrees of glomerulitis and peritubular capillaritis,
(2) frequent TCMR,
(3) both may occur early or late posttransplantation.
However, C4d‑negative AMR morphologically have higher intrarenal endothelial gene expression, alloantibodies expression, poor graft outcomes, usually occur after 1‑year posttransplantation, and associated acute on chronic AMR.
There is another entity of AMR DSA negative AMR, which characterized negative DSA/lack of DSA detection, moderate MVI with (g + ptc) scores of ≥2) as per Banff 2013 with or without C4d positivity.
Accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity, mostly seen in ABO‑incompatible graft transplantation.
Fatima AlTaher
3 years ago
Graft dysfunction results from either TCMD or ABMR ( either C4d + ve or – ve )
In cases of C4d + ve ABMR , complement activation starts after binding of DSA against mismatched HLA with graft endothelium followed by activation of the4 classic complement pathway with endpridction of C4d particles that deposits in PTC and are characteristics for ABMR C4d are positive in following situations
– ABMR
– C ABMR
– ABO incompitable transplant
– Graft accommodation
C4d are – ve in
– False negative due to technical error
– Fc receotors on NK cells mediated rejection
– Non complement fixing ab
– Non HLA Ab
Draw backs of C4d as a marker of rejection
– Interobserver variation in detecting and evaluating the degree of deposition
– Poor reducibilty
Two techniques are used for detection of C4d
in allograft biopsies
1- IF technique : requires frozen sections
– IHC technique requires paraffin‑embedded tissue and more routinely used than IF
For diagnosis of acute or chronic ABMR , C4d stain show
1- With IF , it appears as widespread linear and circumferential PTC staining in both cortex or medulla, excluding areas of scaring or necrotic areas . the significant C4d scoring by IF is >10%
2- In IHC : C4d staining gives linear, continuous, diffuse deposits
3- around in the PTC wall, the significant C4d scoring by IHC is C4d >0
Conclusion
Although C4d is not very sensitive marker of AMR and has several falicies , still its important diagnostic criterion of AMR according to Banff update 2017
Nazik Mahmoud
3 years ago
The Relevance of complement C4d staining in renal allograft Biopsies
Complement is an important component of innate and adaptive immunity in our body,it activated and deposits in renal allograft after the attack of antibodies to the vascular endotheliam.
There’s some conditions showing C4d positive:
Acute AMR
Chronic AMR
ABO incompatible graft
Accommodation
Condition show negative C4d are:
T cell mediated rejection
Technical error
Fc receptor of NK mediated rejection
Ab unable to fix complement
Complement independent pathway of endothelial injury
Very low quantity of C4d to identification by IF or IHC
Allow antibodies directed to endothelium
Increase expression of endothelial transcript causing injury
So C4d detection in PTC has diagnostic and prognostic marker for allograft but had pros like it consider core diagnostic tool to identify AMR and used in many research but the cons were difficult to interpreting focal staining pattern and relatively low sensitive specially in the cases of ABO incompatible transplant
Asmaa Khudhur
3 years ago
Complement 4d (C4d) (that is part of component C4) is a fragment of the classical complement pathway, which is activated by immune complexes. The diagnosis of AMR is improved by identification of the complement pieces C4d in renal biopsy, and it has been introduced first under diagnostic criteria of AMR in year 2003.
clinical relevance Of cOmplement 4d
The conditions where complement 4d is positive
a. Acute AMR
b. Chronic antibody‐mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation.
The state where complement 4d is negative
a. T‐cell‐mediated rejection (TCMR)
b. A technical error like a type of fixatives,
immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the
identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact
with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing endothelium injury.
The C4d‐negative AMR seen with high renal endothelial transcript expression in patients with alloantibody and is an indicator of active antibody‐mediated allograft damage and poor graft outcome.
The C4d‐negative AMR occur due to alloantibodies to MHC molecules on endothelial cells, elicit strong responses that include proliferation and activation of intracellular signaling pathways, which leads to endothelium
antibOdy‐mediated rejectiOn
AMR can occur in three forms, namely hyperacute, acute, and chronic rejection.
The presensitization (blood transfusion, pregnancy, and prior transplant) is a primary risk factor for hyperacute and acute rejection.[17] Acute AMR is most commonly occurs due to expose of MHC Class I and II HLA antigens.[17] Whereas, chronic AMR is largely associated with Class II DSA.
Besides, other antigens which are responsible for AMR included MHC antigen (polymorphic) MICA (MHC Class I‐related chain A) and ABO blood group antigens.
tranSplant glOmerulOpatHy
TG is a variant of chronic AMR and characterized by deposition of C4d.
Morphologically, TG consists of diffuse multilayering or duplication of the glomerular basement membrane (GBM).
cOmplement 4d detectiOn in renal allOgraft
biOpSieS
Two techniques are mostly used for identification of C4d in allograft biopsies of the kidney:
1. Indirect immunofluorescence (IF):
applied to frozen sections
In acute and chronic AMR‐positive C4d stain with IF technique is described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to a consensus at 2003 Banff Conference.
The scoring of C4d is significant by IF on frozen section ie C4d2 or C4d3
C4d is significant staining in 10% or more for frozen/frequency
C4d detection by IF is more sensitive.
Less expensive .
Rapid detection of C4d positivity.
IF method for C4d requires extra tissue and frozen sections facility.
Can not be stored for latter assessment.
Still the gold standard technique
2. Immunohistochemical (IHC):
IHC technique used to paraffin‐embedded tissue used routinely in various laboratories.
C4d staining is crisp, linear, continuous, diffuse, and lying around in the PTC wall, while the strength typically is slighter and changeable, but it may have a finely granular pattern in high power.
The scoring of C4d is significant by IHC C4d >0 on paraffin sections.
C4d detection by IHC more specific
The IHC method for C4d detection feasible in formalin‐fixed, paraffin‐embedded tissue, it is used when frozen sections facility not available, IHC also useful when small biopsy tissue or tissue not available for frozen sections.
The disadvantage of C4d staining by IHC are lower sensibility, nonspecific background staining, more costly and more time consuming, and it needs external controls.
IHC method Widely available.
Uses the same sample that is processed for LM .
The morphology of C4d‐positive and C4d‐negative AMR having following similar features: (1) varying degrees of glomerulitis and peritubular capillaritis, (2) frequent TCMR, (3) both may occur early or late posttransplantation.
There is another entity of AMR DSA negative AMR, which characterized negative DSA/lack of DSA detection, moderate MVI with (g + ptc) scores of ≥2) as per Banff 2013 with or without C4d positivity.
The accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity, mostly seen in ABO‐incompatible graft transplantation.
Antibody-mediated rejection occurs more frequently in individuals who have been sensitized and accounts for 30 per cent of all transplant patients.
A history of exposure to pregnancy or transfusion, as well as a prior transplant, increases the risk of AMR, which can result in hyperacute rejection in most cases.
AMR may be classified into three types: hyperacute acute, acute, and chronic.
acute AMR related with MHC class I and class II exposure b persistent AMR linked with MHC class II exposure
AMR’s pathology is as follows:
glomerulitis, peritubular capillaries, fibrinoid necrosis of arteries, and acute tubular damage are all shown on a microscope in the presence of AMR. Gross enlargement of the kidney owing to interstitial oedema and bleeding is also seen on microscopy.
The presence of DSA and C4d deposits in the PTC is a diagnostic characteristic of AMR.
Causes of C4d positivity include:
. Acute Mycobacterial Respiratory Distress (AMR)
. AMR that is persistent
. Incompatibility with ABO
. T-cell-mediated rejection as a source of C4d negativity as an accommodation (TCMR)
. A technical mistake in the immunofluorescence (IF) or immunohistochemistry (IHC) procedure (IHC)
. Rejection is mediated by the Fc receptor (FCR) on natural killer cells (FcRIIA).
. Antibodies that are incapable of fixing the complement e. Endothelial activation mechanisms are not reliant on the complement.
The presence of C4d-negative AMR due to alloantibodies to MHC molecules on endothelial cells elicits significant reactions that include proliferation and activation of intracellular signalling pathways, which ultimately results in endothelium damage.
In addition, it is present in patients who have developed an alloantibody, and it is a sign of active antibody-mediated allograft destruction and poor graft fate.
AMR causes kidney damage in transplant recipients who are already sensitive. In situations when AMR is left untreated, it can account for up to 30% of all posttransplant rejection episodes, as well as 20%–30% of graft loss after one year after transplantation. AMR classifies rejection into three categories: hyperacute, acute, and chronic.
AMR occurs often in patients who have had a previous blood transfusion, are pregnant, or have had previous transplantation, among other things.
Acute AMR is most usually caused by exposure to MHC Class I and II HLA antigens, which is a common occurrence. Chronic AMR has been shown to be significantly related to Class II DSA.
Hyperacute AMR is characterized as rejection that develops within minutes or within 24 hours of transplantation and is mostly caused by preexisting DSA, among other factors. The graft becomes cyanotic and flaccid on a gross level very quickly. Arteritis, interstitial oedema, and widespread cortical necrosis define the histomorphology of this condition. C4d is accumulated in the PTC and glomerular capillaries of patients with acute AMR. Renal biopsies from patients with chronic AMR show histomorphology defined by transplant glomerulopathy (TG).
Positive staining for C4d in the PTC serves as both a diagnostic and a predictive indication for transplant failure.
Because of the unique properties of C4d, it has (a) provided a tremendous amount of insight into the diagnosis of allograft rejection, (b) served as a core diagnostic tool to identify AMR, and (c) been used for a vast amount of research into the deposition patterns of C4d in different clinical settings such as pregnancy, thrombotic complications, and the following CONS: (1) difficulties in interpreting focal staining patterns, (2) low sensitivity of C4d as a marker for AMR in late renal rejection.
In renal transplant, the allograft is affected by many triggering agents such as innate and adaptive immune mechanisms, either mediated by macrophages and lymphocytes, or by soluble components antibodies and the complement system, which can ultimately lead to graft rejection. Antibody‑mediated rejection (AMR) is a predominant cause of allograft failure. Pathogenesis of Complement 4d
Ag-Ab complexes activate the classical complement pathway with the binding of C4d to the endothelium of peritubular capillaries (PTCS) leading to ABMR
C4d positivity occurs in acute AMR, Chronic antibody‑mediated rejection, ABO incompatible grafts and Accommodation.
C4d negative AMR occurs in T‑cell‑mediated rejection (TCMR),due to technical error in type of fixatives(immunofluorescence (IF) versus immunohistochemistry (IHC), Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection, Antibodies unable to fix the complement, C4d deposition is a very low in quantity for the identification limits of IF/IHC , Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC), Increased expression of endothelial transcripts causing endothelium injury Antibody‑Mediated Rejection: accounts for about 30% of all post-transplant rejection events and 20%–30% graft loss at 1 year if untreated.There are three forms of AMR: hyperacute, acute, & chronic . Acute AMR associated with HLA Class I & II antigens while chronic AMR is associated with Class II DSA. Pathology of AMR: Gross-swollen kidneys(interstitial edema & hemorrhage).Microscopic – glomerulitis, PTCs, fibrinoid necrosis of arteries, & ATN. Other features includepresence of DSA & C4d deposition PTCs. Diagnosis of acute AMR : Diagnosis of acute AMR based on the identification of DSA in plasma, C4 complement component (C4d) in PTC, structural features of AMR, such as kidney injury and graft dysfunction Transplant Glomerulopathy: TG may be one manifestation of humoral rejection of graft injury and consists of diffuse multilayering or duplication of the glomerular basement membrane (GBM) and interstitial fibrosis and atrophy.
Two techniques used for identification of C4d. C4d detection by IHC is more specific & C4d detection by IF is more sensitive .The scoring of C4d is significant by IF on frozen section i.e., C4d2 or C4d3 and by IHC C4d >0 on paraffin sections. C4d & validated transcripts & molecular marker can be potential alternatives & complements to DSAs in the diagnosis of ABMR.
The C4d as a biomarker has following PROS :
(1) provoked an enormous amount of insight in the diagnosis of allograft rejection;
(2) core diagnostic tool to identify AMR;
(3) used for vast amount of research into the deposition patterns of C4d in different clinical settings such as pregnancy, thrombotic complications And has following CONS:
(1) difficulties of interpreting focal staining patterns;
(2) relatively low sensitivity of C4d as a marker for AMR in late renal allograft biopsies;
(3) its lack of utility as a marker for antibody‑mediated injury in biopsies of ABO‑incompatible allografts).
Post kidney transplant, the recognition of the transplanted organ as foreign is mediated by complex immunologic pathways, the Banff criteria divided the rejection in general terms into cellular (T-cell mediated) and humoral (B-cell mediated) pathways.
AMR is a predominant cause of allograft failure. ABMR is caused by antibodies directed against donor-specific human leukocyte antigen (HLA) molecules (DSA) which is considered as a remarkable cause of early and late graft dysfunction.
Complement 4d (C4d) (that is part of component C4) is a fragment of the classical complement pathway, which is activated by immune complexes for diagnosis and identification of the complement pieces C4d biopsy remains the gold standard and provides valuable insightsinto the pathogenesis of early and late allograft injury.
Complement is an important component of innate and adaptive immunity in our body. Complement is the leading effector constituent of adaptive humoral immunity. There are three different pathways of complement activation, immune complex activates the classical pathway when the c1q interplay with immunoglobulin bound to epitopes on the graft endothelium. C1s splits C4 into C4a and C4b and react with exposing a sulfhydryl group giving rise toc4d forming covalent bond with the tissue, C3 convertase (C4bC2a) of classical pathway is formed by combination C4b with enzymatically loose pieces of C2a. it splits C3 into C3a and C3b. C3b has an active sulfhydryl group and forms C5 convertase (C4bC2aC3b). C5 convertase split C5 into C5a and C5b. Further, membrane attack complex (membrane‑bound C5b‑9) formed by the addition of C5b component of C5 convertase, that destroys cells.
C4d positivity can be seen
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. ABO incompatible grafts
d. Accommodation
C4d negativity: can be seen
a. T‑cell‑mediated rejection (TCMR) b. A technical error like a type of fixatives, immunofluorescence (IF) versus immunohistochemistry (IHC) c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection d. Antibodies unable to fix the complement e. Complement independent pathways of endothelial activation f. C4d deposition is a very low in quantity for the identification limits of IF/IHC g. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC) h. Increased expression of endothelial transcripts causing endothelium injury.
The C4d‑negative AMR occur due to alloantibodies to MHC molecules on endothelial cells, elicit strong responses that include proliferation and activation of intracellular signaling pathways, which leads to endothelium injury. The C4d‑negative AMR also seen with high renal endothelial transcript expression in patients with alloantibody and is an indicator of active antibody-mediated allograft damage and poor graft outcome.
AMR can be hyper acute, acute, and chronic rejection. presensitization is a primary risk factor for hyper acute and acute rejection. Acute AMR is most commonly occurring due to expose of MHC Class I and II HLA antigens. Whereas, chronic AMR is largely associated with Class II DSA.
other antigens which has a role in AMR included MHC antigen eg: MICA and ABO blood group antigens.
Histological feature of ABMR – interstitial edema and hemorrhage.
Microscopic findings of AMR- glomerulitis, peritubular capillaritis, fibrinoid necrosis of arteries, and acute tubular injury, Other diagnostic features of AMR requires the presence of DSA and C4d deposition in peritubular capillaries (PTCs).
Hyper acute AMR is defined as rejection occurs within minutes or 24 h after transplantation, mostly due to preexisting DSA present in high titers present in a patient. Grossly, the graft rapidly becomes cyanotic and flaccid. Histomorphology characterized by arteritis, interstitial edema, and extensive cortical necrosis.
In acute AMR, C4d is deposited in PTC and glomerular capillaries.
In chronic AMR, histomorphology characterized by transplant glomerulopathy (TG) in renal biopsies.
TG is mostly seen in humoral rejection TG is a variant of chronic AMR and characterized by deposition of C4d. TG may be one manifestation of humoral rejection of graft injury. Morphologically, TG consists of diffuse multilayering or duplication of the glomerular basement membrane
The C4d‑negative AMR is included in Banff 2013 classification.The morphology of C4d‑positive and C4d‑negative AMR having following similar features: (1) varying degrees of glomerulitis and peritubular capillaritis, (2) frequent TCMR, (3) both may occur early or late posttransplantation.[ However, C4d‑negative AMR morphologically have higher intrarenal endothelial gene expression, alloantibodies expression, poor graft outcomes, usually occur after 1‑year posttransplantation, and associated acute on chronic AMR.
The accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity, mostly seen in ABO‑incompatible graft transplantation.[48] The TMA (thrombotic microangiopathy) is a complication of AMR in the absence of other causes characterized with fibrin thrombi, fragmented red blood cells, mesangiolysis, and muco‑intimal thickening and injury of small vessels.
amiri elaf
3 years ago
III. The Relevance of Complement C4d Staining in Renal Allograft Biopsies
Please summarise this article
# The Banff criteria divided post transplantation rejection in antibody mediated rejection (AMR) and cell mediated rejection.
# AMR is a predominant cause of allograft failure and presence of the DSA consider a remarkable cause of early and late graft dysfunction.
# Complement (C4d) is a fragment of the classical complement pathway, which is activated by immune complexes.
# The diagnosis of AMR is improved by identification of the complement pieces C4d in renal biopsy.
# Complement is an important component of innate and adaptive immunity .
# There are three different pathways of complement activation by macrophages, lymphocytes, soluble components antibodies and the complement system
**classical complement pathway
Activation start with C1 ( C1q, C1r, and C1s)
C1q with immunoglobulin bound to epitopes on the graft endothelium. C1s splits C4 into C4a and C4b.Then C3 convertase (C4bC2a) splits C3 into C3a and C3b. C3b forms C5 convertase (C4bC2aC3b).
C5 convertase split C5 into C5a and C5b. followed by membrane attack complex (membrane bound C5b‑9)
**The lectin pathway of complement activates by Mannan binding lectin (MBL) , H‑ficolin or L‑ficolin.
The MBL attach to the suitable carbohydrate on apoptotic cells or pathogens.
** Alternative pathway
# The following conditions are showing C4d positivity:
* Acute AMR
* Chronic antibody mediated rejection
* ABO incompatible grafts
* Accommodation.
# Following conditions are showing C4d negativity:
*Tcell mediated rejection
* Technical error like a type of fixatives ,
immunofluorescence IF versus immunohistochemistry IHC
* Fc receptor on NK cells (FcRIIA) mediated
rejection
* Antibodies unable to fix the complement
* Complement independent pathways of endothelial activation
* C4d deposition is a very low in quantity for the identification limits of IF/IHC
* Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC)
* Increased expression of endothelial transcripts causing endothelium injury
# Antibody mediated rejection
# The AMR harms allograft kidney in sensitized patients, it accounts for up to 30% of all post transplant rejection and 20%–30% graft loss at 1 year in untreated cases.
# AMR Three types, hyper acute, acute, and chronic rejection.
The pre sensitization is a primary risk factor for hyper acute and acute rejection.
* Hyperacute AMR is defined as rejection occurs within minutes or 24 h after transplantation, mostly due to preexisting DSA present in high titres , the graft rapidly becomes cyanotic and flaccid, histomorphology characterized by arteritis, interstitial edema, and extensive
cortical necrosis.
*Acute AMR is most commonly associated with MHC class 1and II DSA
# Other antigen responsible for AMR included MICA (MHC Class I‑related chain A) and ABO blood group antigens
# Microscopic findings include glomerulitis, peritubular capillaritis, fibrinoid necrosis of arteries, and acute tubular injury also presence of DSA and C4d deposition in peritubular capillaries and glomerular capillaries leading to kidney injury and graft dysfunction
* Chronic AMR is largely associated with class II DSA.
There is glomerulopathy (TG) in renal biopsies, as capillary basement membrane duplication or splitting and mesangial expansion of glomeruli, tubular atrophy interstitial fibrosis, and fibrous intimal thickening in arteries and peritubular capillary basement membrane multilayering.
# The effector mechanisms of AMR:
* Direct effects of antibody to MHC
* Complement fixation
* Cellular FCRs
* Mixed cellular and humoral rejection are very hard occurs after months and years posttransplantation, and
biopsy show acute and chronic lesion.
# Transplant Glomerulopathy
TG is a morphological change that is mostly seen in humoral rejection and related to immunological etiology, is a variant of chronic AMR and characterized by deposition of C4d, diffuse multilayering or duplication of the glomerular basement membrane their lack distinct de novo or repetitive glomerular lesion or TMA
# Transplant Glomerulopathy Also Associated
Multilayering of Peritubular Capillarie
There is arterial intimal fibrosis of new onset, leukocytic
infiltration in intima indcating chronic rejection with TG.
TG was connected with the diagnosis of AMR of either peritubular capillaritis, glomerulitis or both as well as with C4d
expulsion in PTCs.
# Complement 4d Detection in Renal Allograft Biopsies C4d detected by using:
*IHC on paraffin embedded tissue; (crisp, linear, continuous, diffuse, and lying around in the PTC wall).
* IF applied to frozen sections (widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic area).
# Current Status of Complement 4d
Devadass et al. reported C4d is not very sensitive marker of
AMR
Banff update 2017 both C4d and validated transcripts/classifiers/molecular marker can serve as potential alternatives and complements to DSAs in the diagnosis of ABMR.
# Complement 4d Negative Antibody Mediated Rejection
The morphology of C4d positive and C4d negative AMR having following similar features:
* Varying degrees of glomerulitis and peritubular capillaritis *Frequent TCMR
* Both may occur early or late posttransplantation. C4d negative AMR morphologically have higher intrarenal endothelial gene, alloantibodies expression& poor graft outcomes occur after 1 year posttransplantation and associated acute on chronic AMR.
The accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity,
mostly seen in ABO incompatible graft transplantation.
# Conclusion
The C4d as a biomarker has following PROS
* Provoked an enormous amount of insight in the diagnosis of allograft rejection
* Core diagnostic tool to identify AMR
* Used for vast amount of research into the deposition patterns of C4d in different clinical settings
CONS of C4d
* Difficulties of interpreting focal staining
Patterns
*Relatively low sensitivity of C4d as a marker
for AMR in late renal allograft biopsies
*Its lack of utility as a marker for antibody mediated injury in biopsies of ABO incompatible allografts
Introduction: Acute rejection is divided into Antibody-mediated rejection and cell-mediated rejection according to Banff’s classification C4d is a fragment of a classical complement pathway which helps in the diagnosis of antibody-mediated rejection by detection of C4d Clinical importance of positive C4d : Acute AMR
Chronic AMR
Incompatible ABO kidney transplantations
Accommodation Antibody‑Mediated Rejection may be hyperacute, acute, and chronic, responsible for 20-30 % of graft loss in first-year post-transplantation, and associated with DSA against Class I and Class II of HLA antigens or DSA to non-HLA antigens like (MICAand ABO blood antigens) classified as : Hyperacute rejection: with first 24 hours (cyanotic graft and cortical necrosis) Acute : glomerulitis and peritubular capiliritis Chronic: transplant glomerulopathy Mixed Antibody and cellular: after months or years and biopsy may show acute or chronic affections Transplant glomerulopathy: pathology includes: subendothelial broadening, interstitial fibrosis, tubular atrophy, and intimal fibrosis of arteries C4d detection in Biopsies : IF C4d 2 or 3
IHC > 0
essential for diagnosis of acute ABM however decrease sensitivity in chronic ABM C4d negative ABM rejection: maybe associated with acute or chronic ABM rejection with higher endothelial gene expression, Alloantibodies with poor graft survival C4d positive with no ABM rejection in ABO incompatible transplantation with accommodation TMA BK nephropathy pregnancy Conclusion C4d as PROS : 1-has role in the detection of allograft rejection 2-diagnostic item in ABM rejection 3-detected in pregnancy and thrombotic events C4d as CONS : 1-low sensitivity marker in ABM in late biopsies
2-difficult interpretations of C4d
3- decrease importance in ABO-incompatible allograft biopsies with antibody-mediated injury
Renal allograft is affected by many triggering agents such as innate and adaptive immunity which can lead to graft rejection. ABMR is the predominant cause of allograft failure, and DSA is a remarkable cause of early and late graft dysfunction. There is more development about C4d after inclusion in the diagnostic criteria of ABMR.
Clinical Relevance of C4d:
Positive C4d is seen in:
acute ABMR
chronic ABMR
ABO incompatible transplant
accomodation
Negative C4d seen in:
TCMR
technical errors
Fc receptor on NK cells mediated rejection
complement independent pathway of endothelial activation
C4d deposition is very low in quality for the identification limits of IF/IHC
alloantibodies can direct endothelium injury to interact with MHC
increased expression of endothelial transcripts causing endothelium injury
ABMR:
accounts for 30% of all post transplant rejection events and 20-30% graft loss at 1 year in untreated cases.
ABMR can occur in 3 forms: hyper acute, acute, and chronic. Presensitization is the risk factor for hyperacute and acute rejection.
Pathological findings of ABMR is grossly swollen kidney due to odema and hemorrhage. Microscopic findings include glomerulitis, peritubular capillaritis, fibrinoid necrosis of arteries and acute tubular injury. Other diagnostic features are the presence of DSA and C4 d deposition in the peri tubular capillaries.
hyperacute rejection occurs within minutes to 24 hr post transplant due to preexisting DSA, acute rejection C4d is deposited in the ptc and glomerular capillaries while chronic ABMR is characterized by the presence of TG.
The effector mechanisms of ABMR are:
direct effect of antibody on MHC
complement fixing
cellular FCR
diagnosis of ABMR is based on the identification of DSA in plasma, C4d in ptc, and graft dysfunction.
TG:
mostly seen in humoral rejection related to immunological injury, characterized by C4d deposition. It consists of multilayering of GBM. IF and IHC show C4d deposition along the GBM presented in the minority of cases (6% in one series).
TG is also associated with multilayering of peri tubular capillaries:
TG was connected with the diagnosis of ABMR in the existence of either peri tubular capillaritis or glomerulitis or both as well as with C4d expulsion in PTCs.
C4d detection in allograft biopsies:
two techniques are used for identification of C4d, they are IHC and IF.
In ABMR, positive C4d staining with IF technique is described as widespread, strong linear circumferential ptc staining in cortex or medulla, excluding scar area according to Banff 2013.
In IHC C4d staining is crisp, linear, continuous, diffuse, and lying around in the ptc wall, it’s strength is slightler and changeable but it may have a fine granular pattern in high power.
The scoring of C4d is significant by IF on frozen section ( C4d2, C4d3) and by IHC C4d> 0 in paraffin sections.
The DSA is most specifically associated with C4d deposition in ptc and it’s interactivity with endothelial cells in the graft.
Current state of C4d:
studies showed that C4d is not very sensitive marker of rejection as it was thought originally. Currently C4d has become an essential investigation for ABMR according to Banff 2017. In this update, both C4d and validated transcripts/ classifiers/ molecular markers can serve as potential alternatives and complements to DSA in the diagnosis of ABMR.
C4d negative ABMR:
It was included in the Banff 2013 classification, has similar morphology to C4d positive rejection, but have higher intrarenal endothelial gene expression, alloantibody expression, poor graft outcomes, usually occur after 1 year post transplantation and associated with acute and chronic ABMR.
Complement is an important component of innate and adaptive immunity in our body. Complement is the leading effector constituent of adaptive humoral immunity. There are three different pathways of complement activation and their activator in kidney tranplantion.
The AMR harms allograft kidney, chiefly in more sensitized patients. AMR accounts for up to 30% of all posttransplant rejection events and 20%–30% graft loss at 1 year in untreated cases.
AMR can occur in three forms, namely hyperacute, acute, and chronic rejection .
Acute AMR is most commonly occurs due to expose of MHC Class I and II HLA antigens,Whereas, chronic AMR is largely associated with Class II DSA .
Types of ABMR,
a-Hyperacute AMR,rarely seen now adays
b-Acute AMR,
C4d positive in peritubular capillarities and glomerular capillarities
c-Chronic AMR:
characters by transplant glomerulopathy manifested by capillary basement membrane duplication and mesangial expansion of glomeruli, tubular atrophy and interstitial fibrosis and fibrous intimal thicking.
Complement 4d Detection in Renal Allograft Biopsies,
Two techniques are mostly used for identification of C4d IHC and IF in allograft biopsies of the kidney. IF technique applied to frozen sections whereas IHC technique used to paraffin embedded tissue used routinely in various laboratories.
Clincal relevance of complement C4d
Following conditions are showing C4d positivity ,
Acute AMR
Chronic antibody mediated rejection
A, B, O blood group ABO incompatible grafts
4- Accommodation.
Following conditions are shown c4d negative,
1. Tcellmediated rejection (TCMR)
. 2 .A technical error like a type of fixatives, immunofluorescence (IF) versus immunohistochemistry (IHC)
3. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
4. Antibodies unable to fix the complement
5. Complement independent pathways of endothelial activation
6. C4d deposition is a very low in quantity for the identification limits of IF/IHC
7. Alloantibodies can direct endothelium injury to interact with major
histocompatibility complex (MHC)
8. Increased expression of endothelial transcripts causing endothelium injury.
What is Transplant Glomerulopathy?:
Morphologically, TG consists of diffuse multilayering or duplication of the glomerular basement membrane (GBM).
A variant of chronic AMR
Deposition of C4d in a minority of cases.
Connected to diagnosis of AMR, in the existence of either PTCs or glomerulitis or both as well as with C4d in PTCs.
What is c4d negative antibody mediated rejection?
C4d negative AMR morphologically have higher intrarenal endothelial gene expression, alloantibodies expression, poor graft outcomes, usually occur after 1 year posttransplantation, and associated acute on chronic AMR.
The morphology of C4dpositive and C4dnegative AMR having following similar features:
varying degrees of glomerulitis and peritubular capillaritis,
frequent TCMR
oth may occur early or late posttransplantation
Conclusion,
The C4d positive staining in PTC is a diagnostic marker as well as a prognostic marker for allograft.
C4d appears to be a less sensitive marker than initially thought.
The detection of AMR should be best reported based on morphological features such as tubulointerstitial, vascular, and glomerular histological changes, with a piece of legislation to the presence or absence of C4d. Despite all pitfalls C4d is excellent marker for AMR
Last edited 3 years ago by MOHAMMED GAFAR medi913911@gmail.com
SUMMARY OF The Relevance of Complement C4d Staining in Renal Allograft BiopsiesRenal allograft affect by immune response either mediated by macrophages and lymphocytes or by soluble component (antibodies and complement system) which can lead to graft rejection
AMR is a predominant cause of allograft failure
DSA to HLA antigens cause earlier and late graft failure
C4d is afragment of classical complement pathway which is activated by antigen antibody complex
presence of c4d renal biopsy improve diagnosis of AMR Pathogensis of C4d:
activation of complement of 3 different pathways
complement classical pathway is initiated when c1q with immunoglobulin bind to epitope on the graft endothelium
C1s split c4 into c4a and c4b and then c4b to c4d which is covalent bind to tissue Clinical of c4d +ve conditions:
1) acute AMR
2) chronic AMR
3) ABO incompatible graft
4) A commendation C4d -ve status in the following conditions:
TCMR
A technical error eg type of fixation IF versus IHC
FC receptor (FCR) on NK cells (FCR11A) mediated rejection
Antibody unable to fix the complement
complement independent pathway of endothelial activation
c4d is very low in quantity for the identify of IF-IHC
alloantibody can direct endothelium injury to interact with MHC
increase expression of endothelial transcript causes endothelial injury
Antibody mediated rejection:
AMR represent 30% of all post transplant rejection event and 20-30% graft loss at 1 year
3 forms of AMR include hyper acute ,acute,and chronic rejection
The primary risk factor of AMR (hyper acute and acute rejection) is presensitization
MHC class 1&2 HLA antigens associated with acute AMR
MHC class 2 DSA is associated with AMR
MHC antigen polymorphism MICA (MHC class1 related chain A) and ABO blood group antigen all these antigens are responsible for AMR
To diagnose AMR require :
pathological finding in form of glomerulitis pertubular cappilaritis fibrinoid necrosis of arteries and acute tubular injury
presence of DSA in the patients serum
PTCs c4d deposition
Hyper acute AMR:
Occur with minutes to hours post renal transplant
High titre of DSA in patients
histomorphological: artaritis .interstitial edema and extensive cortical necrosis
Chronic AMR:
Transplant glomerulopathy
tubular atrophy and interstitial fibrosis
Mechanism of AMR:
direct effect of antibody to MHC
complement fixation
cellular FCRs
Mixed cellular and humeral rejection:
Hard to examine
occur in months or years post transplant
histological feature of acute and chronic changes
AMR is the main cause of graft damage when IHC for c4d in PTC with concomitant presence of DSA in the serum than cellular rejection
Transplant glomerulopathy:
Is a chronic form of AMR characterized by
multi layering or duplication of the GBM
multi layering of peritubular capilleries
C4d deposition in GBM in 6% of cases
Similarity of morphology of c4d +ve and c4d -ve in form of:
glomerulitis and peritubular capillaritis
frequent TCMR
both may occur earlier or late post transplant
C4d -ve AMR associated with:
increase endothelial gene expression
alloantibodies expression
poor graft outcome
occur after 1 year post transplant
associated acute on chronic AMR
accommodation definition:
c4d deposition in PTC in the absence of active rejection with or without DSA ,seen in ABO incompatible graft Conclusion:
C4d as biomarker has following PROS:
provoke and enormous amount of insight in the diagnosis of allograft rejection
diagnostic tool to identify AMR
Used in reaserch into the deposition pattern in different setting(pregnant and thrombotic complications)
CONS of c4d as biomarker as follows:
difficult of interpretating focal staining pattern
In late renal allograft biopsy has low sensitivity as marker for ABMR
It lack of utility as marker for antibody mediated injury in biopsy of ABO incompatible graft
Despite all pitfalls c4d is excellent marker for ABMR
1.C4d is a foot print of Classical Complement System activation
2. C4d establishes a covalent bond with the tissues and gets deposited for a reasonably long duration in tissues
3. C4d can be the result of activation by Classical pathway, Lectin Pathway and also by C-reactive protein
4. Conditions where c4d can be deposited:
– ABMR – Both Acute & Chronic
– ABO incompatible grafts
– Accommodation 5. Conditions where C4d is Negative:
– TCMR
– False negativity due to technical issues
– NK cell mediated Rejection
– Non Complement Fixing DSA
– Endothelial activation due to Non complement mediated mechanisms
– C4d present but below the threshold of deposition
– Presence of increased expression of Endothelial Transcripts- A marker of poor graft outcome
– Alloantibodies directly interacting with MHC molecules on the endothelium
6. Acute AMR = Exposure of Class 1 & 2Antigens
Chronic AMR = Exposure of Class 2 Antigens 7. C4d Detection in Renal Allograft Biopsies:
– Two Techniques
– IF on Frozen Sections —> Widespread, Strong, Linear Circumferential
– IHC on Paraffin Sections —> Crisp, Linear, Circumferential, Linear with variable intensity
– C4d is significant staining in 1% (Any degree) or more of the PTC for formalin/IHC‐IP, or 10% or more for frozen/frequency IF 8. Prospects of C4d:
– C4d in addition to validated transcripts/classifiers/molecular marker can become potential alternatives or may complement DSA 9. Complement Negative Antibody Mediated Rejection:
– C4d negative AMR was included in 2013 Banff
– How C4d -ve ABMR differs from C4d +ve ABMR: Higher infrarenal endothelial gene expression, Alloantibody expression, Poor Graft Outcomes, Usually after 1 yr post Transplant, Association with Acute on Chronic AMR 10. What is Accommodation:
– C4d deposition in PTCs / absence of Active rejection / DSA +/-
– usually seen in ABOi Tx 11. How much intensity of C4d is Significant ?
– What Banff 2019 States: C4d, linear staining in PTCs or medullary vasa recta by immunofluorescence (IF) on frozen sections of fresh tissue or immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded tissue, scored as 0 (none), 1 (minimal, staining in >0 but <10% of PTCs), 2 (focal, 10%-50% of PTCs), 3 (diffuse, >50% of PTCs). By IF on frozen sections scores ≥2 are considered positive; by IHC on paraffin sections all scores >0 are considered positive
– To simplify, IHC : Any degree of staining ; By IF : > 50 % of staining
– C4d detection by IHC more specific and C4d detection by IF is more sensitive 12. IF Vs IHC:
– IHC: More Specific / Paraffin embedded Tissue / Small Biopsy tissue also Sufficient / Time Consuming / Non specific background Staining
– IF: More sensitive / Frozen sections / Require extra tissue 13. “Pinch of Salt” Facts about C4d: ?? Not as sensitive marker of ABMR as thought initially / Can not be used in ABOi Transplant setting
III. The Relevance of Complement C4d Staining in Renal Allograft Biopsies Please summarise this article Innate & adaptive immune systems, mediated by cellular (macrophages & lymphocytes) or soluble components (antibodies & complement system), affect graft function & can lead to rejection. AMR is a predominant cause of allograft failure. Anti-HLA DSA are significant cause of early & late graft dysfunction & failure. C4d is a fragment of the classical complement pathway, that is activated by antigen/antibody complexes. Diagnosis of AMR improves by detection of C4d in renal biopsy. This review evaluates pathogenesis & current relevance of C4d in AMR. Pathogenesis of C4d: Complement is the major constituent of adaptive humoral immunity. Clinical Relevance of C4d C4d positivity is seen in: a. Acute AMR b. Chronic AMR c. ABOi grafts d. Accommodation. C4d negativity is seen in: a. TCMR b. A technical error e.g. type of fixatives, IF versus IHC c. Antibodies unable to fix the complement d. Complement independent endothelial activation e. C4d deposition is very low to be detectable by IF/IHC f. Alloantibodies to MHC on endothelial cells leading to endothelial injury. g. Increased expression of endothelial transcripts causing endothelium injury. Antibody‑Mediated Rejection
– 30% of all post-transplant rejection events
– 20%–30% graft loss at 1 year if untreated.
– Occur in 3 forms: hyperacute, acute, & chronic
– Presensitization is a primary risk factor for hyperacute & acute rejection.
– Acute AMR associated with HLA Class I & II antigens.
– Chronic AMR is associated with Class II DSA.
– MICA & ABO antigens are other causes of AMR. Pathology of AMR: Gross-swollen kidneys(interstitial edema & hemorrhage). Microscopic – glomerulitis, PTCs, fibrinoid necrosis of arteries, & ATN. Presence of DSA & C4d deposition PTCs. Chronic-TG in renal biopsies. Diagnosis of acute AMR Based on DSA in plasma, C4d in PTC, clinical & structural features of AMR. AMR is the chief cause of graft damage specially with positive C4d in PTC combined with high levels of DSA. Minimum or absent c4d positivity in PTC & absent DSA in plasma make AMR less likely. Transplant Glomerulopathy: – A variant of chronic AMR – Deposition of C4d in a minority of cases. – Connected to diagnosis of AMR, in the existence of either PTCs or glomerulitis or both as well as with C4d in PTCs. C4d in allograft Biopsies C4d score is significant by IF if C4d2 or C4d3 & by IHC C4d >0. C4d detection by IHC is more specific & C4d detection by IF is more sensitive. Current Status of C4d Poor inter-observer reproducibility for C4d IHC An essential test for AMR(Banff update 2017). C4d & validated transcripts & molecular marker can be potential alternatives & complements to DSAs in the diagnosis of ABMR. C4d-negative AMR Included in Banff 2013. C4d-positive & C4d-negative AMR have some similarities: – Varying degrees of glomerulitis – Peritubular capillaritis – Frequent TCMR – Both may occur early or late post-transplantation. – C4d‑negative AMR have higher intrarenal endothelial gene expression, alloantibodies expression, & poor graft outcomes. DSA negative AMR: Characterized by: – Lack of DSA detection – Moderate MVI with (g + ptc) scores of ≥2(Banff 2013) – With or without C4d positivity. The accommodation: Characterized by: – C4d deposition in PTCs – Absence of active rejection – With or without DSA positivity – Seen in ABOi transplantation. Conclusion PTC C4d-positive staining in PTC is a diagnostic as well as a prognostic marker for allograft rejection. However, from many studies, C4d appears to be a less sensitive marker than initially thought. Some cons of C4d as a biomarker include: – Difficulty of interpreting focal staining patterns – Relatively low sensitivity as a marker for AMR in late renal allograft biopsies – Its lack of utility as a marker for AMR in ABOi allograft biopsies. The detection of AMR should best focus on histological features without much reliance on the presence or absence of C4d. However C4d is still an excellent marker for AMR.
Post-transplant graft rejection can happen either due to cellular or antibody-mediated immunological causes. Banff criteria classify rejection into 2 main types, namely antibody mediated rejection (AMR) and cell-mediated rejection (TCMR). AMR caused by donor specific antibodies (DSA) is a predominant cause of graft failure.
Immune complexes activate classical complement pathway, cleaving C4 into C4a and C4b, which in turn bind to hydroxyl or amino group containing molecules via sulfhydryl group giving rise to C4d forming a covalent bond with the tissue. C4 can also be activated through lectin pathway by MBL, H-ficolin and L-ficolin.
C4d positivity is seen in acute AMR, chronic AMR, ABO incompatible transplant and accommodation (C4d deposition in peritubular capillaries without rejection, with or without DSAs).
Rejection with C4d negative stain can be seen with a TCMR, technical error (due to fixatives, IF or IHC), FcRIIA (Fc receptor on NK cell) mediated rejection, non-complement fixing antibodies, very low quantity C4d deposits (below the identification limit of IF/IHC), in conditions with antibodies causing direct endothelial injury (non-HLA antibodies) and with endothelial injury due to increased expression of endothelial transcripts.
AMR (hyperacute, acute or chronic) involves 30% of all rejections and 20-30% of graft loss at 1 year in untreated cases. Past history of sensitization is associated with hyperacute and acute rejection. Acute AMR is associated with both class I and II DSA while chronic AMR is mainly associated with class II DSA. Other antibodies associated with AMR include antibodies against ABO blood group antigens and MICA.
Characteristics of AMR include interstitial edema and hemorrhage causing swollen graft with glomerulits, peritubular capillaritis, fibrinoid necrosis and acute tubular injury, C4d deposition in peritubular capillaries with presence of DSA.
Hyperacute rejection presents within minutes to hours, with a cyanotic and flaccid graft having interstitial edema and widespread cortical necrosis. Acute AMR has C4d in PTC and glomerular capillaries. Chronic AMR shows transplant glomerulopathy (TG).
TG consists of capillary basement membrane splitting or duplication, mesangial expansion of glomeruli, interstitial fibrosis, tubular atrophy and fibrous intimal thickening in arteries, with C4d deposition and without evidence of de novo or recurrent glomerular lesions and TMA. C4d deposition is less in TG.
C4d staining can be done either using immunofluorescence (IF) of frozen sections (significant if ≥10%) or immunohistochemistry (IHC) on formalin preserved paraffin embedded tissue (significant if ≥1%). It has been shown that C4d is not a very sensitive marker for AMR. Microvascular inflammation (MVI) correlated with diffuse C4d positive cases. In Banff 2017 update, for a diagnosis of AMR, in addition to DSA and acute tissue injury, either C4d or transcripts/classifiers/ molecular markers can be used.
Although C4d negative AMR has characteristics similar to C4d positive AMR (frequent TCMR, may occur early or late, varying degree of glomerulitis and peritubular capillaritis), it usually occurs late, has increased alloantibody expression with increased intrarenal endothelial gene expression, is associated with poor graft outcomes due to increased acute on chronic AMR.
C4d is a marker for AMR but has low sensitivity in late graft rejection and is not useful in ABO incompatible transplants. So it is imperative that AMR be reported on the basis of histomorphological features with C4d staining complimenting the biopsy findings.
The Relevance of Complement C4d Staining in Renal Allograft Biopsies Introdution Post kidney transplant, the allograft is affected by many stimulating agents such as antigen‑ antibody‑mediated immunocomplexes and cellular immunity mediated by various cells (macrophages and lymphocytes), which could lead to graft rejection. Based on etiology, the Banff criteria divided the rejection broadly in antibody‑mediated rejection (AMR), and cell‑mediated rejection. AMR is a predominant cause of allograft failure. The clinicians and pathologists now consider antibodies against human leukocyte antigen (HLA) antigens defined as donor‑specific antibodies (DSA) a remarkable cause of early and late graft dysfunction Complement 4d (C4d) (that is part of component C4) is a fragment of the classical complement pathway, which is activated by immune complexes. The diagnosis of AMR is improved by identification of the complement pieces C4d in renal biopsy, and it has been introduced first under diagnostic criteria of AMR in year 2003.
Pathogenesis of complemente 4d Complement is an important component of innate and adaptive immunity in our body. Complement is the leading effector constituent of adaptive humoral immunity. There are three different pathways of complement activation, culminating in the deposit of C4d: – Classical: – Alternative: – Lecitin:
Antibody Mediated Rejection
The AMR harms allograft kidney, chiefly in more sensitized patients. AMR accounts for up to 30% of all posttransplant rejection events and 20%–30% graft loss at 1 year in untreated cases. AMR can occur in three forms, namely :
– Hyperacute: The presensitization (blood transfusion, pregnancy, and prior transplant) is a primary risk fator. Occurs within minutes or 24 h after transplantation, mostly due to preexisting DSA present in high titres present in a patient. Grossly, the graft rapidly becomes cyanotic and flaccid. Histomorphology characterized by arteritis, interstitial edema, and extensive cortical necrosis
– Acute: The presensitization (blood transfusion, pregnancy, and prior transplant) is a primary risk factor and acute AMR is most commonly occurs due to expose of MHC Class I and II HLA antigens. C4d is deposited in PTC and glomerular capillaries.
– Chronic rejection: is largely associated with Class II DAS. Histomorphology characterized by transplant glomerulopathy (TG) in renal biopsies. The TG specified as capillary basement membrane duplication or splitting and mesangial expansion of glomeruli, tubular atrophy and interstitial fibrosis, and fibrous intimal thickening in arteries
Diagnosis of acute AMR based on the identification of DSA in plasma, C4 complement component (C4d) in PTC, structural corroboration of AMR, such as kidney injury and graft dysfunction. Mixed cellular and humoral rejection are very hard to examine because it mostly occurs after months and years posttransplantation, and biopsy may show acute and chronic lesion. Here, it is tough to prove that cellular or humoral immune system responsible for the extracellular injury.
Complement 4d Detection in Renal Allograft Biopsies
Two techniques are mostly used for identification of C4d IHC and IF in allograft biopsies of the kidney. IF technique applied to frozen sections whereas IHC technique used to paraffin‑embedded tissue used routinely in various laboratories.
Current Status of Complement 4d
Devadass et al. reported C4d is not very sensitive marker of AMR, as it originally thought. Histologically, moderate to marked microvascular inflammation (MVI) correlated with dispersing C4d positive cases in contrast to focal C4d‑positive cases.
Currently, C4d has become an essential investigation for AMR according to Banff update 2017. In this update, both C4d and validated transcripts/classifiers/molecular marker can serve as potential alternatives and complements to DSAs in the diagnosis of ABMR.
The C4d‑negative AMR is included in Banff 2013 classification. The morphology of C4d‑positive and C4d‑negative AMR having following similar features, however, C4d‑negative AMR morphologically have higher intrarenal endothelial gene expression, alloantibodies expression, poor graft outcomes, usually occur after 1‑year posttransplantation, and associated acute on chronic AMR.
The accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity, mostly seen in ABO‑incompatible graft transplantation.
The TMA (thrombotic microangiopathy) is a complication of AMR in the absence of other causes characterized with fibrin thrombi, fragmented red blood cells, mesangiolysis, and muco‑intimal thickening and injury of small vessels.
Conclusion
The C4d‑positive staining in PTC is a diagnostic marker as well as a prognostic marker for allograft. According to various studies worldwide, C4d appears to be a less sensitive marker than initially thought. The C4d as a biomarker has following PROS :
– (1) provoked an enormous amount of insight in the diagnosis of allograft rejection;
– (2) core diagnostic tool to identify AMR;
– (3) used for vast amount of research into the deposition patterns of C4d in different clinical settings such as pregnancy, thrombotic complications
And has following CONS:
– (1) difficulties of interpreting focal staining patterns;
– (2) relatively low sensitivity of C4d as a marker for AMR in late renal allograft biopsies;
– (3) its lack of utility as a marker for antibody‑mediated injury in biopsies of ABO‑incompatible allografts).
Despite all pitfalls C4d is excellent marker for AMR.
Antibody mediated rejection occur more in sensitised patients and account 30% of all transplant patients.
History of exposure to pregnancy or transfusion and previous transplant are risk for AMR and usually lead to hyperacute rejection.
Forms of AMR are hyperacute
acute and chronic AMR.
acute AMR due to exposure to MHC class I and class II
chronic AMR associated with exposure to class II DSA.
Pathology of AMR:
grossly swelling of kidney due to interstitial edema and hemorrhage
microscopy finding of AMR shows a. glomerulitis
b. peritubular capillarities
c. fibrinoid necrosis of arteries
d. acute tubular injury
Diagnostic feature of AMR is presence of DSA and C4d deposits in PTC
Causes of C4d positive:
1. acute AMR
2. chronic AMR
3. ABO incompatible
4. Accommodation
causes of C4d negative:
T‐cell‐mediated rejection (TCMR)
b. A technical error of
immunofluorescence (IF) v
or immunohistochemistry
(IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity
g. Alloantibodies can direct endothelium injury to interact
with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing
endothelium injury.
Hyperacute AMR:
Rejection within minutes to 24 hours and occur due to presence of DSA
Grossly graft cyanotic and flaccid
Histomorphological shows arteritis, interstitial edema and extensive cortical necrosis.
Acute AMR:
C4d positive in peritubular capillarities and glomerular capillarities
Chronic AMR:
characters by transplant glomerulopathy manifested by capillary basement membrane duplication and mesangial expansion of glomeruli, tubular atrophy and interstitial fibrosis and fibrous intimal thicking.
Diagnosis of AMR:
1. presence of circulating DSA
2. C4d in peritubular capillary
Mixed cellular and humoral are very hard to examine and occur months to years post transplant in biopsy and may show acute & chronic lesions.
Transplant glomerulopathy:
it’s humoral allograft rejection characteristics by deposition of C4d in peritubular capillarities.
histomorphological manifested by duplication of basement membrane due to accumulation of mesangial cell.
C4d detected by immunoflorecence and immunohistochemistry
by immunoflorescence shows strong linear circumferential peritubular capillary staining in cortex and medulla.
in immunohistochemistry C4d staining linear continuous and diffuse and lying around PTC.
Circulating DSA are associated with C4d positive.
Current status of complement C4d is diagnostic marker for AMR according to banff classification 2017 and associated with moderate to severe micro vascular inflammation.
C4d negative AMR have higher intrarenal endothelial gene expression, alloantibodies and poor graft outcome after one year and associated with acute & chronic AMR.
Accommodation: C4d positive in PTC in absence of acute AMR with or without DSA and seen in patients with ABO incompatible.
Thrombotic microangiopathy is complication of AMR and manifested by fibrin thrombi and fragments RBC and mesangiolysis and muco intimal thickning and vessels injury.
Conclusion:
C4d positive is diagnostic and prognostic marker for AMR but not sensitive.
The Relevance of Complement C4d Staining in Renal Allograft Biopsies This review aims to evaluate pathogenesis and current relevance of C4d in AMR
C4d is a degradation product of the classic complement pathway. After an antigen-antibody complex fixes complement, a cascade of events follows with activation of several complement proteins. The complement protein C4 is split into C4a and C4b. C4b is then converted to C4d.
C4d is a complement fragment that is generated through classic pathway activation and is covalently bound to antigen. The results of several studies suggest that peritubular capillary C4d deposition is a marker for acute humoral rejection.
diagnosis of AMR is improved by identification of the complement pieces c4d in renal biopsy, and it has been introduced first under diagnostic criteria of AMR.
Complement 4d is positive in the following conditions.
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation.
Complement 4d is negative in the following situations
a. T‑cell‑mediated rejection (TCMR)
b. A t e c h n ic a l e r r o r l i ke a t y p e of f i x a t ive s ,immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing endothelium injury C4d‑negative AMR due to alloantibodies to MHC molecules on endothelial cells, elicit strong responses that include proliferation and activation of intracellular signaling pathways, which leads to endothelium injury.
Also seen in in patients with alloantibody and is an indicator of active antibody‑mediated allograft damage and poor graft outcome.
AMR harms allograft kidney, more sensitized patients. AMR accounts for up to 30% of all posttransplant rejection events and 20%–30% graft loss at 1 year in untreated cases. AMR grades hyperacute, acute, and chronic rejection.
AMR occurs widely in presenstized patient whom previously have blood transfusion, pregnancy ,previous transplantation .
Acute AMR is most commonly occurs due to expose of MHC Class I and II HLA antigens. chronic AMR is largely associated with Class II DSA.
Hyperacute AMR is defined as rejection occurs within minutes or 24 h after transplantation, mostly due to preexisting DSA, Grossly, the graft rapidly becomes cyanotic and flaccid. Histomorphology characterized by arteritis, interstitial edema, and extensive cortical necrosis , In acute AMR, C4d is deposited in PTC
and glomerular capillaries. In chronic AMR, histomorphology characterized by transplant glomerulopathy (TG) in renal biopsies. Conclusion
C4d‑positive staining in PTC is a diagnostic marker as well as a prognostic marker for allograft.
C4d properties (1) provoked an enormous amount of insight in the diagnosis of allograft rejection, (2) core diagnostic tool to identify AMR, and (3) used for vast amount of research into the deposition patterns of C4d in different clinical settings such as pregnancy, thrombotic complications and CONS as follows: (1) difficulties of interpreting focal staining patterns, (2) relatively low sensitivity of C4d as a marker for AMR in late renal allograft biopsies, and (3) its lack of utility as a marker for antibody‑mediated injury in biopsies of ABO‑incompatible allografts.
The Relevance of Complement C4d Staining in Renal Allograft Biopsies. Introduction:
This review aims to evaluate pathogenesis and current relevance of C4d in AMR.
C4d is considered criterion for diagnosis ABMR since 2003,which is degradation of complement factor 4 which is apart from classical complement pathway which activated by immune-complex and stays at the site of complement activation for a longer time, in comparison to other complement pieces. Pathogenesis of Complement 4d:
When immunoglobulin bounds to epitopes on the graft endothelium , classic complement pathway start to be activated via C1q then C1s splits C4 into C4a and C4b and react with exposing a sulfhydryl group. The ester or amide bond forms with interacting between sulfhydryl group of C4b and nearby molecules containing hydroxyl or amino groups in the tissue.
Cascade become activated with many steps to reach finally to membrane attack complex (membrane‑bound C5b‑9) formed by the addition of C5b component of C5 convertase, that destroys cells.(figure .1) Clinical Relevance of Complement 4d :
The conditions where complement 4d is positive are:
a. Acute AMR.
b. Chronic antibody‑mediated rejection.
c. A, B, O blood group ABO incompatible grafts.
d. Accommodation. The state where complement 4d is negative:
a. T‑cell‑mediated rejection (TCMR).
b. A technical error like a type of fixatives,
immunofluorescence (IF) versus immunohistochemistry
(IHC).
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated
rejection.
d. Antibodies unable to fix the complement.
e. Complement independent pathways of endothelial
activation.
f. C4d deposition is a very low in quantity for the
identification limits of IF/IHC.
g. Alloantibodies can direct endothelium injury to interact
with major histocompatibility complex (MHC).
h. Increased expression of endothelial transcripts causing
endothelium injury. Antibody‑Mediated Rejection:
Acute AMR is most commonly occurs due to expose of MHC Class I and II HLA antigens.
But chronic AMR is largely associated with Class II DSA.
Acute ABMR diagnosed by histological changes(MVI, glomerulitis ,C4d positive and DSA).
Chronic ABMR characterized by chronic histological changes like TG which is duplication of GBM. Transplant Glomerulopathy:
Histological description which is duplication of the glomerular basement membrane (GBM).
Interstitial fibrosis and tubular atrophy existed along with TG.
Complement 4d Detection in Renal Allograft Biopsies:
Detected by IF or IHC , the first one is more sensitive than IHC and IHC is more specific. Current Status of Complement 4d:
Currently, C4d has become an essential investigation for AMR according to Banff update 2017.
Complement 4d Negative Antibody‑Mediated Rejection:
The C4d‑negative AMR is included in Banff 2013 classification with same morphological changes of ABMR but no stain for c4d less worse than c4d positive , but also has bad effect on graft survival.
Another form described by BANFF 2013 is DSA negative ABMR with or without c4d. Conclusion:
C4D is a great marker for ABMR, it has advantages e.g. core diagnostic tool to identify AMR and dis advantages e.g. difficulties of interpreting focal staining patterns and relatively low sensitivity of C4d as a marker for AMR in late renal allograft biopsies , but still is considered diagnostic marker as well as a prognostic marker for allograft.
The aim of this review study to determine the pathogenesis and the clinical applicability of C4d.
Introduction: Kidney transplantation can be hampered by allograft rejection in particular AMR due to the effect of the antibodies against HLA antigens as well as cellular immune response. C4D is the split product of the classical complement pathway activation by antibodies -antigen immune response during allograft rejection it’s one of the Banff diagnostic criteria of the AMR since 2003 Clinical applicability of C4D C4D positive
1- Acute and chronic ABMR
2- ABO i Transplantation
3- accommodation of allograft with or without DSA and not associated with allograft injury or rejection
4- Autoimmune diseases like SLE, MGN, MPGN, Ig A nephropathy C4D negative:
1-Acute cellular rejection (TCMR)
2- technical error by using IF vs IHC staining, IF more sensitive while IHC more specific for C4D staining, also false negative with low level of C4D for detection.
3- presence of non-complement fixing DSA, non-HLA abs
4- complement in dependent pathway of rejection, cytotoxic T cell activation, cellular immune response like in C4D negative ABMR
5- FC NKC receptor induced AR
6-endothilial injury transcription expression which indicate endothelitits in C4 D negative ABMR injury, ENDAT which is carry poor prognosis and lead to progressive graft loss.
Antibody mediated rejection:
The prevalence of AMR up to 30%, and can lead to graft loss in 20-30% after 1year of transplantation, many types of AR including hyper-acute rejection, acute AMR and chronic AMR, acute AMR it due to the performed anti HLA class1 DSA, while chronic AMR is due to class11 anti-HLA DSA.
Histologically AMR characterized by C4D deposition in PTCS, glomerulitis and acute tubular injuries while in Chronic AMR characterized by transplant glomerulopathy (Tg) with the histological features of glomerular capillary basement membrane duplication and splitting, mesangial widening with IFTA, and by EM typical finding of PTC multilayering, chronic intimal arterial thickening and fibrosis
Diagnosis of AMR need the following:
Presence of PTCS, glomerulitis, IHC staining for c4d in addition to the circulatory DSA and clinical evidence of acute graft dysfunction The effector mechanism of AMR
Direct effect of the DSA on the MHC,
Complement activation (classical complement pathway) with the production of the C4D split product consider the footprint of the antibody’s antigen interaction
Endothelitis with microvascular endothelial injury by cellular FCR NKC.
C4D detection by either using immunohistochemically (IHC) on formalin embedded paraffin sections with positive score >0, and 1% or more positive c4d staining of the PTC the IHC Staining is more specific but less sensitive compared to IF staining while the scoring of C4d is significant if 10% and above by IF on frozen section.
still C4d staining is part of the investigation for AMR
according to Banff update 2017.[44]
but recently with the improvement of the molecular genetic testing both C4d
and validated molecular markers of endothelial transcripts/ molecular marker are essential in the diagnosis of AMR including c4d negative non-complement mediated AMR. Conclusion:
the C4d‑positive staining in PTC is still an indicative and prognostic marker
for allograft dysfunction and still essential part for the diagnosis of AMR in combination with the typical histological morphology but keep in mind the lower sensitivity of C4D as a marker for AMR in late renal allograft biopsies (non-complement mediated AMR, also in ABO I-transplantation with accommodation and this can be resolved by adding the new molecular genetic biomarkers .
This article elaborates on different aspects of AMR pathogenesis and relevance of C4d.
AMR is the predominant cause of allograf failure. Antibodies to human leukocyte antigen( HLA)
defined as DSAs are considered a remarkable cause of early and late graft dysfunction.
C4d is part of complement component C4 which is a fragment of classical complement pathway.
Classical complent pathway is activated by immune complexes. C4d make covalent bond with tissue.
It stay at the site of complement activation for a longer time than the other components.
Conditions associated with C4d positivity:
1) Acute AMR
2) Chronic AMR
3) A,B.O incompatible grafts
4) Accommodation.
Negative C4d staining conditions:
1) CMR
2)techn8cal error IF vs IHC.
3) Cytotoxic dependent AMR
4)Complement independent pathway of endothelial activation.
5)Increased expression of endothelial transcripts causing endothelial injury.
AMR:
Account for 30% of all post transplant rejection.
AMR is classified into 3 categories,hyperacute, acute and chronic.
Hyperacute AMR ,
presensitization due to blood transfusion,pregnancy and prior transplantation is an essential risk factor. It occurs within minutes to 24 HOURS, features interstitial edema, artritis and extensive cortical necrosis
Acute AMR :
is due to DSAs against Class I and class II HLA antigens. With activation of complement and deposition of C4d in PTCs and glomerular capillaries.
Chronic AMR :
is due to anti class II DSAs.characterized by TG which features duplication and splitting of GBM,Mesangial expantion IFTA, and fibrous intimal thickening. C4d deposition along GBM presented in minority of cases (6% in one series). It’s associaated with PTC multilayring detected by EM. Glomerulitis and peritubular cap8llaritis are another 2 connection to TG.
Non HLA antigens involved in AMR include
1)MICA(MHC Class I related chain A).
2)ABO blood group antigens.
Effector mechanisms of AMR:
1)Direct effect of DSAs on HLA .
2)Complement fix8ng DSAs.
3)Cellular FCRs.
Diagnostic Criteria of AMR:
1-Microscopic finding of glomerulitis,peritubular capillaritis PTC,fibrinoid necrosis of arteries,and Acute tubular injury.
2-presence of DSAs.
3-C4d deposition in PTCs. Detected by IF or IHC.
Mixed CMR and AMR:
Hard to diagnose, because it occurs months or years post transplantation.
C4d vs MVI:
MVI emerged as another feature reliable to consider in diagnosing AMR.C4d and validated transcript/classifier/molecular marker can serve as potential alternative and complement to DSAs in diagnosing AMR.
C4d negative AMR:
1)Both can occur early or late.
2)vary8ng degree of PTC inflammation,
3)C4d negative AMR have higher intra-renal gene expression,alloantibodies expression,poor graft outcome,and associated with acute on chronic AMR.On the contrary,DSAs negative AMR, ,characterized by absence of DSAs with MVI. And positive or negative C4d.
Anti HLA DSA are now considered by pathologists and clinicians the culprit of early and late graft injury and failure. C4d is a degradation product of the classical complement pathway, which is activated by antigen antibody complexes.
Complement is an important component of innate and adaptive immunity in our body. Complement system activation is the major contributor of allograft injury in antibody mediated rejection.
The complement system can be activated through three major pathways: classical, lectin, and alternative.
Classical pathway is activated when the C1 complex (C1q, C1r and C1s serine proteasess), binds to the Fc region of complement-fixing antibodies attached to the antigen. Activation of C1r and C1s in turn cleaves C4 and C2 into larger (C4b, C2a) and smaller (C4a, C2b) fragments. This activity underpins the entire complement system terminating in the production of anaphylatoxins and assembly of the MAC and resulting in target cell damage/lysis.
Clinical Relevance of Complement 4d
Following conditions are showing C4d positivity:
a. Acute AMR
b. Chronic antibody mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation.
The state where complement 4d is negative
a. T cell mediated rejection
b. A technical error like a type of fixatives, IF versus IHC
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with MHC
h. Increased expression of endothelial transcripts causing endothelium injury.
AMR can occur in three forms, namely hyperacute, acute, and chronic rejection. Presensitization is a primary risk factor for hyperacute and acute rejection. The diagnosis of AMR requires th
Transplant Glomerulopathy is a morphological change that is mostly seen in humoral rejection and related to immunological etiology. TG is a variant of chronic AMR and characterized by deposition of C4d.
Ultra structure features of TG consist of cytoplasmic vacuolation, broadening of subendothelial space, with the existence of electron dense floccular material, probably due to stiffening of the lamina rara interna. Interstitial fibrosis and tubular atrophy existed along with TG.
Two techniques are mostly used for identification of C4d IHC and IF in allograft biopsies of the kidney. IF technique applied to frozen sections whereas IHC technique used to paraffin embedded tissue.
Currently, C4d has become an essential investigation for AMR according to Banff update 2017.
C4d staining of the kidney biopsy is important in the diagnosis of renal pathology .
C4d staining can be positive in the following conditions :
Active and chronic active antibody mediated rejection : the sensitivity of C4d staining in these conditions is around 50 % ( 50% of antibody mediated rejection is C4d negative ) .
ABO incompatible transplantation.
In states of accommodation : mainly occur in ABO incompatible transplantation in which there is diffuse staining for C4d with no evidence of tissue injury or DSA , such patients had good graft survival nearly equal to patients with ABO compatible transplantation .
In antibody mediated rejection the C4d staining maybe negative due to non-complement binding antibodies ( in which the graft damage is due to antibody mediated cellular cytotoxicity or direct damage to endothelial cells by the anti HLA antibodies ) or due to low quantity of the C4d deposition that not detected by the stain.
C4d staining can be done with either IF or IHC , IF is more sensitive and the results is rapid , whereas IHC is more specific and can be done in cases when no frozen tissue is available but more costly and time consuming
ABMR : is predominant causes of allograft failure .
DSA a remarkable cause of early and late graft dysfunction .
C4d is fragment of the classical complment pathway , which is activated by Ag _Ab complexes. the dignosis of ABMR by detection of the complement fragment C4d in renal biopsy.
the conditions are showing c4d +ve
acute ABMR
chronic ABMR
A,B, O blood group ABO incompatiable grafts
accommodation
C4d -ve
T -cell mediated rejection
techical error
Fc receptors on NKcell mediated rejection
complement independent pathway of endothial activation
alloantibodies can direct endothilum injury to interact withMHC
increased expression of endothial transcripts causing endothial injury
C4d deposition is avery low in quantity for identification limitsof IF \ IHC
C4d -ve associted with poor graft outcome.
ABMR : occuring in three forms hyperacute , acute and chronic AMR and most commonly occurs due to expose of MHC class 1 and 2
HLA and chronic ccuring with class 2 DSA
pathological finding ABMR kideny become swollen due to interstitial edema and hemmorrage
Microscopic finding of ABMR glomerulitis ,peritubular capillaritis , fibrinoid necrosis of arteries and acute tubular injury.
and present DSA with C4d deposition in peritubular capillaries.Hyperacute AMR occurs within minutes or 24 h after transplantation, caused by preexisting DSA in high titres .Pathological wise characterized by arteritis, interstitial edema, and extensive cortical necrosis. Acute AMR C4d is deposited in PTC and glomerular capillaries. Chronic AMR pathologicaly associated with transplant glomerulopathy (TG) specified as capillary basement membrane duplication or splitting and mesangial expansion of glomeruli, tubular atrophy and interstitial fibrosis, and fibrous intimal thickening in arteries. AMR mechanism :antibodies directed against MHC, Complement fixation, and Cellular FCRs. mixed cellular and humoral rejection are very hard to examine beacuse it mo0stly occuring after months and years postransplantion transplant glomerulopathy
Is a morphological change that is mostly seen in humoral rejection and related to immunological etiology and it is a variant of chronic AMR and characterized by deposition of C4d.
TG consists of diffuse multilayering or duplication of the glomerular basement membrane which best seen by jones methenamine silver and periodic acid‑Schiff stain.
Ultra‑structure features of TG consist of cytoplasmic vacuolation, broadening of subendothelial space, with the existence of electron‑dense floccular material.
Interstitial fibrosis and tubular atrophy existed along with TG.
complement c4d detection in renal allograft biopies acute and chronic AMR‑positive C4d stain with IF technique is demonstrated as widespread, strong linear circumferential PTC staining in cortex or medulla, apart from scar or necrotic areas as stated by Banff 2003. In IHC, C4d staining is crisp, linear, continuous, diffuse, around in the PTC wall, while the strength is slighter , but can have a finely granular pattern in high power. current status of complement 4d currently C4d has become an essential investigation for ABMR according to banff update 2017
complement c4d -ve ABMR
c4d -ve ABMR and + ve ABMR having similar features glomerulitis and peritubular capillaritis , frequent TCMR Both occur early or late posttransplation note c4d -ve have higher interarenal endothial gene expression with poor graft outcomes. the accomodation is defined as c4d deposition in PTC in absence of active rejection with or without DSA +ve and mostly seen in ABO incompatible . TMA is a complication of ABMR in abscence other cause ( fragmented RBC , mesangiolysis and muco intimal thicking and injury of small vessels . conclusion C4d staining in PTC is a diagnostic marker .and prognostic marker for marker for allograft.
.
Complement 4d (C4d) is a fragment of the classical complement pathway, which is activated by immune complexes . C4d makes covalent bond with the tissue. It stays at the site of complement activation for a longer time, in comparison to other complement pieces.
Pathogenesis of complement 4d ;
In the classical pathway of complement kick over of C1 is begun by an interplay of C1q with immunoglobulin bound to epitopes on the graft endothelium. C1s splits C4 into C4a and C4b and react with exposing a sulfhydryl group. C3 convertase (C4bC2a) of classical pathway is formed by combination C4b with enzymatically loose pieces of C2a. C4bC2a (C3 convertase) splits C3 into C3a and C3b. C3b has an active sulfhydryl group which is dative bound and settles in the immediate locality and forms C5 convertase (C4bC2aC3b). C5 convertase split C5 into C5a and C5b. Further, membrane attack complex (membrane‑bound C5b‑9) formed by the addition of C5b component of C5 convertase, that destroys cells.
Following conditions are showing C4d positivity:
a. Acute AMR
b. Chronic AMR
c. ABO incompatible grafts
d. Accommodation.
The following conditions are showing C4d negativity:
a. (TCMR).
b. A technical err or like a type of fixative .
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection.
d. Antibodies unable to fix the complement.
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with major. histocompatibility complex (MHC) .
h. Increased expression of endothelial transcripts causing endothelium injury
Anti body mediated rejection ;
The effector mechanisms of AMR;
(1) Direct effects of antibody to MHC .
(2) Complement fixation .
(3) Cellular FCRs.
The DSA produced by plasma cells bind to the endothelium of donor PTC and glomerular capillaries and start the pathological progression of AMR. The C1q is a component of the complement of the classical pathway which ties to the endothelium‑binding DSA, finally leading to graft injury and dysfunction. Diagnosis of acute AMR based on the identification of DSA in plasma, C4 complement component (C4d) in PTC, structural corroboration of AMR, such as kidney injury and graft dysfunction.
Transplant glomerulopathy ;
TG is a variant of chronic AMR and characterized by deposition of C4d. Morphologically, TG consists of diffuse multilayering or duplication of the glomerular basement membrane (GBM).
Complement 4d detection in renal allograft biopsy ;
Two techniques are mostly used for identification of C4d IHC and IF in allograft biopsies of the kidney.
IF technique;
applied to frozen sections . In acute and chronic AMR‑positive C4d stain with IF technique is described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to a consensus at 2003 Banff Conference. C4d detection by IF is more sensitive.
IHC technique;
used to paraffin‑embedded tissue used routinely in various laboratories. In IHC, C4d staining is crisp, linear, continuous, diffuse, and lying around in the PTC wall, while the strength typically is slighter and changeable, but it may have a finely granular pattern in high power. C4d detection by IHC more specific
Complement 4d negative anti body –mediated rejection ;
C4d‑negative AMR morphologically have higher intrarenal endothelial gene
expression, alloantibodies expression, poor graft outcomes, usually occur after 1‑year post transplantation, and associated acute on chronic AMR.
There is another entity of AMR DSA negative AMR, which characterized negative DSA/lack of DSA detection, moderate MVI with (g + ptc) scores of ≥2) as per Banff 2013 with or without C4d positivity.
The accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity, mostly seen in ABO‑ incompatible graft transplantation.
Conclusion ;
The C4d‑positive staining in PTC is a diagnostic marker as well as a prognostic marker for allograft. According to various studies worldwide, C4d appears to be a less sensitive marker than initially thought.
C4d is a complement component ( product of C4) which is activated by antigen-antibody interaction
The diagnosis AMR is facilitated by incorporation C4d in the allograft biopsy in Banff 2013 meeting[6]
The objective of this review is to determine the pathogenesis & the significance of C4d in AMR.
Pathogenesis ;
Complement-dependent mechanism
C4 is cleaved by C1 into C4a & C4b ; C4d has a sulfhydryl group rapidly interact with amide or ester bond of the surrounding molecules having hydroxyl or amino groups anf forms C4d[4]
C4 forms covalent bonds with endothelial cells[5]
Clinical relevance of C4d ; Conditions associated with +ve C4d ; [11,12]
Acute AMR
Chronic AMR
ABO- incompatible grafts
Accommodation = C4d +ve with or without DSA in ABO-incompatible transplant with no evidence of AMR
Causes of C4d -ve staining ;[10-14]
TCMR
Technical error
C4d amount is below the threshold for detection
Complement-independent mechanism
Antibodies are unable to fix complement
Increase expression of endothelial transcript
AMR ;
20 -30% of graft loss at 1 year if not treated[15]
Sensitization is the major risk factors e.g pregnancy, transfusion, prior transplant[17]
Acute occurs due to both classI & class II antibodies, while chronic AMR mostly due to class II
In acute AMR , grossly the graft is swollen due to interstitial edema & hemorrhages. Histologic findings are glomerulolitis / peritubular capillaritis, ATI, intimal arteritis[22]. Other findings are DSA in the serum , +ve C4d in the biopsy[23]
AMR causes graft rejection through direct injury or resentments of the inflammatory cells or complement-dependent mechanism[26,27]
TG ;
A form of chronic AMR[2,23]
Main findings are ; 1. GBM duplication(new basement membrane formation) in absence of TMA 2. ptc multi-layering 3.C4d deposition[35,37] 4. arterial intimal fibrosis[11]
C4 detection in allograft biopsies ; 2 methods ; linear diffuse deposition[6]
IF on frozen-section
IHC on parafin-embedded section
Current status of C4d ;
Important for diagnosis of AMR
At least 10% by IF & any degree of positivity by IHC[43]
C4d negative AMR ;
Identified by Feucht et al., & Edmond group[45]
Associated with high expression of the endothelial transcript[46]
Important cause of both early & late graft dysfunction[11]
Conclusion ;
C4d is important but less sensitive marker for allograft rejections[52]
High expression of endothelial transcript on biopsy may be useful in cases of C4d -ve[46]
C4d is a component of classical complemet pathway which activated by immune complexes. C4d has become an essential investigation for AMR according to Banff update 2017
C4d positive in the following conditions
1. Acute AMR.
2. Chronic antibody‑mediated rejection.
3. A, B, O blood group ABO incompatible grafts.
4. Accommodation.
C4d negative in the following conditions.
1. T‑cell‑mediated rejection (TCMR)
2. A technical error.
3. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection.
4. Antibodies unable to fix the complement.
5. Complement independent pathways of endothelial activation.
6. C4d deposition is a very low in quantity for the identification limits of IF/IHC.
7. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC).
8. Increased expression of endothelial transcripts causing endothelium injury
In acute AMR, C4d is deposited in peritubular capillaries and glomerular capillaries. The diagnosis of acute AMR involves positive DSA in plasma, C4 complement component (C4d) in peritubular capillaries and structural corroboration of AMR. Transplant glomerulopathy is a variant of chronic AMR and characterized by deposition of C4d.
In conclusion, C4d‑positive staining in peritubular capillaries is a diagnostic and prognosis marker for renal allograft. The advantages for c4d include assist in diagnosis of allograft rejection, core diagnostic tool to identify AMR, and used in research into the deposition patterns of C4d in different clinical settings such as pregnancy, thrombotic complications. The disadvantages include difficulties of interpreting focal staining patterns, relatively low sensitivity of C4d as a marker for AMR in late renal allograft biopsies, and less clinical relevant as marker for antibody‑mediated injury in biopsies of ABO‑incompatible allograft.
RELEVANCE OF COMPLEMENT C4D STAINING IN RENAL ALLOGRAFT BIOPSIES
SUMMARY
Complement 4d (C4d) (that is part of component C4) is a fragment of the classical complement pathway, which is activated by immune complexes.C4 is cleaved by C1s into C4a and C4b, exposing a sulfhydryl group. The active sulfhydryl group of C4b rapidly forms an amide or ester bond with nearby molecules containing hydroxyl or amino groups and forms C4d .
C4d positivity:
a. Acute AMR b. Chronic antibody‑mediated rejection c. A, B, O blood group ABO incompatible grafts d. Accommodation.
complement 4d is negative :
a. T‑cell‑mediated rejection (TCMR)
b. A technical error like a type of fixatives, immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing endothelium injury.
Two techniques are mostly used for identification of C4d IHC and IF in allograft biopsies of the kidney.
The scoring of C4d is significant by IF on frozen section ie C4d2 or C4d3 and by IHC C4d >0 on paraffin sections.
The circulating DSA are most specifically associated with C4d deposition in PTC and its interactivity with endothelial cells in the graft.
The accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity, mostly seen in ABO‑incompatible graft transplantation.
Despite all pitfalls C4d is excellent marker for AMR.
Post kidney transplant, the allograft is affected by many stimulating agents such as antigen‑ antibody‑mediated immunocomplexes and cellular immunity mediated by various cells (macrophages and lymphocytes), which could lead to graft rejection.
-Complement is an important component of innate and adaptive immunity in our body. Complement is the leading effector constituent of adaptive humoral immunity.
-Following conditions are showing C4d positivity:
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation.
*The state where complement 4d is negative
The following conditions are showing C4d negativity:[10‑14]
a. T‑cell‑mediated rejection (TCMR)
b. A technical er ror li ke a type of fixatives,
immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing
endothelium injury
*The C4d‑positive staining in PTC is a diagnostic marker as well as a prognostic marker for allograft. According to various studies worldwide, C4d appears to be a less sensitive marker than initially thought.The C4d as a biomarker has following
PROS (1) provoked an enormous amount of insight in the diagnosis of allograft rejection, (2) core diagnostic tool to identify AMR, and (3) used for vast amount of research into the deposition patterns of C4d in different clinical settings
such as pregnancy, thrombotic complications and CONS as follows: (1) difficulties of interpreting focal staining patterns, (2) relatively low sensitivity of C4d as a marker
for AMR in late renal allograft biopsies, and (3) its lack of utility as a marker for antibody‑mediated injury in biopsies of ABO‑incompatible allografts). However, in which cases C4d not helpful in diagnosis whereas molecular studies have furnished perceptiveness evocative of a complement‑independent form of AMR or C4d‑negative AMR. According to us, the detection of AMR should be best reported based on morphological features such as tubulointerstitial, vascular, and glomerular histological
changes, with a piece of legislation to the presence or absence of C4d. Despite all pitfalls C4d is excellent marker for AMR.
References
1. Nankivell BJ, Alexander SI. Rejection of the kidney allograft. N Engl J Med2010;363:1451‑62.
2. Mengel M, Sis B, Haas M, Colvin RB, Halloran PF, Racusen LC, et al. Banff 2011 Meeting report: New concepts in antibody‑mediated rejection. Am J Transplant2012;12:563‑70.
3. Tait BD, Süsal C, Gebel HM, Nickerson PW, Zachary AA, Claas FH, et al. Consensus guidelines on the testing and clinical management issues associated with HLA and non‑HLA antibodies in transplantation. Transplantation 2013;95:19‑47.
4. Monsinjon T, Gasque P, Chan P, Ischenko A, Brady JJ, Fontaine MC. Regulation by complement C3a and C5a anaphylatoxins of cytokine production in human umbilical vein endothelial cells. FASEB J 2003;17:1003‑14.
5. Chakravarti DN, Campbell RD, Porter RR. The chemical structure of the C4d fragment of the human complement component C4. Mol Immunol 1987;24:1187‑97.
6. Racusen LC, Colvin RB, Solez K, Mihatsch MJ, Halloran PF, Campbell PM, et al. Antibody‑mediated rejection criteria‑An addition to the Banff 97 classification of renal allograft rejection. Am J Transplant 2003;3:708‑14.
7. Magil AB, Tinckam K. Monocytes and peritubular capillary C4d deposition in acute renal allograft rejection. Kidney Int 2003;63:1888‑93.
AMR due to DSA antigens is an important cause of graft loss. C4d in renal biopsy demonstrates classic complement pathway activation by Ag – Ab complexes. C1q Splits C4 to C4a and C4b to and then forms C3 convertase (C4b C2a) to split C3. There reactions finally form C5 convertase (C4b C2a C3b), that splits C5 to C5a and C5b and forms MAC. Other ways for complement activations are lectin and alternative pathways. C4d staining is positive in:
1) Acute ABMR 2) Chronic ABMR 3) ABOi 4) Accommodation:
There are 8 conditions which are C4d negative:
1) TCMR
2) Technical errors
3) FCR II A on NK cells causing rejection
4) non-complement fixing Abs
5) Very low deposition of C4d
6) Allo-Abs directly cause endothelium injury
7) Endothelia activation independent of complement pathways.
8) Expression of endothelial transcripts.
Sensitized patients are at risk of AMR. There forms of AMR are hyper-acute, acute and chronic. Acute form is related to MHC class I and II and chronic form is associated with class II. Other Ags like MICA, MICB and ABO antigens are involved in same cases.
Transplant glomerulopathy means GBM duplication or multilayering. It may be associated with peritubular capillaries multilayering. C4d staining by IHC or IF on allograft biopsies in PTC shows evidence for circulating DSA. According to Banff 2017 both C4d and transcript and are alternative to DSA.
Accommodation means C4d deposition in PTCs without active rejection or positive DSA and is seen in ABOi TX.
C4d detection by IHC is specific but its detection by IF is more sensitive. Nowadays there are pros and cons about the importance of C4d as a biomarker for graft.
C4d is one of diagnostic criteria of antibody mediated rejection.
It is a fragment of the classical complement pathway, makes covalent bond with the tissue and remain at site of complement activation for long time.
Complement is part of adaptive humoral immunity.
It is activated by 3 pathways, the classical pathway, the lectin pathway and may be activated by CRP.
Clinical relevance of C4d
Conditions were C4d is positive:
Acute antibody mediated rejection
Chronic antibody mediated rejection
ABO blood group incompatible grafts
Accommodation
Conditions with C4d negative:
T-cell mediated rejection
Fc receptor on NK cells mediated rejection
Technical error
Antibodies unable to fix complement
Complement independent pathway of endothelial activationC4d is low in quantity for Identification limits
Increased expression of endothelial transcripts causing endothelial injury.
In acute AMR, C4d is deposited in peritubular and glomerular capillaries.
Chronic AMR is characterized by transplant glomerulopathy in renal biopsy
The effector mechanism of AMR include direct effect of antibodies to MHC, complement fixation and cellular FCRs.
Diagnosis of AMR requires detection of DSA in serum, deposition of C4d in PTC and histological evidence of AMR
Absence of C4d deposition in PTC and DSA in serum decrease the probability of AMR.
Transplant glomerulopathy:
C4d in biopsies:
Immunohistochemistry and immunofixation are used in identification of C4d.
IF is applied to frozen sections, C4d stain is described as widespread, strong linear circumferential PTC staining, C4d2 or C4d3 is significant score
while IHC is applied to paraffin embedded tissue used routinely in various labs, C4d staining is crisp, linear and diffuse deposition in PTC wall, C4d>0 is significant score.
IF is more sensitive while IHC is more specific, used when frozen section is not available.
C4d is not very sensitive marker for AMR.
Interobserver variation of IHC staining is improved with the binary scoring system.
According to Banff 217, C4d is an essential investigation for AMR
C4d and validated transcripts are potential alternatives to DSA in diagnosis of AMR.
C4d negative AMR:
have higher antibody expression, poor graft outcome, usually occur after one year, may be acute or chronic and have increased intrarenal endothelial gene expression.
Accommodation is defined as C4d deposition in PTC in absence of active rejection with or without DSA seen in ABO incompatible transplant.
C4d staining is diagnostic and prognostic marker
Pros:
Used in diagnosis of AMR
Used in research in deposition patterns of C4d in different clinical settings.
Cons:
Difficulties in interpreting focal staining patterns
low sensitivity
Can’t be used as marker for AMR in ABO incompatible transplant
The Relevance of Complement C4d Staining in Renal Allograft Biopsies
Complement 4d (C4d) is a fragment of the classical complement pathway (that is a part of component C4), which is activated by antigen‑antibody complexes. The diagnosis of AMR improves by detection of the complement fragment C4d in renal biopsy, and it has included for diagnosis of AMR in the year 2003. There is more development about C4d after inclusion in the diagnostic criteria of AMR. This
review aims to evaluate pathogenesis and current relevance of C4d in AMR.
Pathogenesis of complement 4d (complement system and complement 4d)
There are three different pathways of complement activation and their activator
The conditions where complement 4d is positive
Following conditions are showing C4d positivity
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation.
The state where complement 4d is negative
The following conditions are showing C4d negativity:[10‑14]
a. T‑cell‑mediated rejection (TCMR)
b. A technical error like a type of fixatives, immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing endothelium injury
The C4d‑negative AMR occur due to alloantibodies to MHC molecules on endothelial cells, elicit strong responses that include proliferation and activation of intracellular signaling pathways, which leads to endothelium injury. The C4d‑negative AMR also seen with high renal endothelial transcript expression in patients with alloantibody and is an indicator of active antibody‑mediated allograft damage and poor graft outcome.
Antibody‑mediated rejection
AMR can occur in three forms, namely hyperacute, acute, and chronic rejection.
The presensitization (blood transfusion, pregnancy, and prior transplant) is a primary risk
factor for hyperacute and acute rejection.
Acute AMR is most commonly occurring due to expose of MHC Class I and II HLA antigens. Whereas, chronic AMR is largely associated with Class II DSA.
Complement 4d detection in renal allograft biopsies
Two techniques are mostly used for identification of C4d IHC and IF in allograft biopsies of the kidney.
Current status Of complement 4d
C4d is not very sensitive marker of AMR, as it originally thought. Histologically, moderate to marked microvascular inflammation (MVI) correlated with dispersing C4d positive cases in contrast to focal C4d‑positive cases.
Currently, C4d has become an essential investigation for AMR according to Banff update 2017
Conclusion
The C4d‑positive staining in PTC is a diagnostic marker as well as a prognostic marker for allograft. According to various studies worldwide, C4d appears to be a less sensitive marker
than initially thought.
The C4d as a biomarker has following PROS (1) provoked an enormous amount of insight in the
diagnosis of allograft rejection, (2) core diagnostic tool to identify AMR, and (3) used for vast amount of research into the deposition patterns of C4d in different clinical settings such as pregnancy, thrombotic complications
and CONS as follows: (1) difficulties of interpreting focal staining patterns, (2) relatively low sensitivity of C4d as a marker for AMR in late renal allograft biopsies, and (3) its lack of
utility as a marker for antibody‑mediated injury in biopsies of ABO‑incompatible allografts)
Despite all pitfalls, C4d is an excellent marker for AMR.
In case of mixed rejection graft loss most likely due to Ab mediated rejection rather than T .cell mediated rejection .
what I learn from this paper that what previously called suspicion Ab mediated rejection(absence of DSA with histopathological picture of Ab mediated rejection ) now could be confirmed as Ab mediated rejection if biopsy confirmed positive PTC4d and intr renal endothelial gene expression .
Introduction
the allograft is affected by many stimulating agents such as antigen‑ antibody‑mediated immunocomplexes and cellular immunity mediated by various cells (macrophages and lymphocytes), which could lead to graft rejection, Complement 4d (C4d) (that is part of component C4) is a fragment of the classical complement pathway, which is activated by immune complexes.
The conditions where complement 4d is positive:
a. Acute AMR.
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation.
The state where complement 4d is negative:
a. T‑cell‑mediated rejection (TCMR)
b. A technical error like a type of fixatives, immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing endothelium injury.
AMR accounts for up to 30% of all posttransplant rejection events and 20%–30% graft loss at 1 year in untreated cases.
Acute AMR is most commonly occurs due to expose of MHC Class I and II HLA antigens. Whereas, chronic AMR is largely associated with Class II DSA.
There suggested that AMR is the chief cause of graft damage, which positive IHC for C4d in PTC in the combination of high levels of DSA in the serum than cellular rejection.
Transplant glomerulopathy TG: is a morphological change that is mostly seen in humoral rejection and related to immunological etiology.TG is a variant of chronic AMR and characterized by deposition of C4d.
C4d is not very sensitive marker of AMR, as it originally thought. Histologically, moderate to marked microvascular inflammation (MVI) correlated with dispersing C4d positive cases in contrast to focal C4d‑positive cases.
C4d is significant staining in 1% or more of the PTC for formalin/IHC‑IP, or 10% or more for frozen/frequency IF.
Currently, C4d has become an essential investigation for AMR according to Banff update 2017. In this update, both C4d and validated transcripts/classifiers/molecular marker can
serve as potential alternatives and complements to DSAs in the diagnosis of ABMR.
The morphology of C4d‑positive and C4d‑negative AMR having following similar features: (1) varying degrees of glomerulitis and peritubular capillaritis, frequent TCMR, (3) both may occur early or late posttransplantation,However, C4d‑negative AMR morphologically have higher intrarenal endothelial gene expression, alloantibodies expression, poor graft outcomes, usually occur after 1‑year post transplantation, and associated acute on chronic AMR.
According Banff diagnostic criteria, C4d staining in PTCs (at least 10% PTC positive is considered as significant) by IHC on paraffin sections Banff score C4d > 0 is significant. In contrast, by IFs on frozen sections Banff scores C4d2 or C4d3 is considered significant .Thus, it is indicated that C4d detection by IHC more specific and C4d detection by IF is more sensitive.
The IHC method for C4d detection feasible in formalin‑fixed, paraffin‑embedded tissue, it is used when frozen sections facility not available, IHC also useful when small biopsy tissue or tissue not available for frozen sections.The disadvantage of C4d staining by IHC are lower sensibility, nonspecific background staining, more costly and more time consuming, and it needs external controls. IF method for C4d requires extra tissue and frozen sections facility
The Relevance of Complement C4d Staining in Renal Allograft Biopsies
Post-transplant Allograft is affected by many stimuli including antibody-mediated immunocomplex and cellular immunity. Antibody-mediated rejection (AMR) is a major cause of graft loss. The clinicians and pathologists now define antibodies against HLA antigens (DSA) as a remarkable cause of early and late graft dysfunction.
C4 is cleaved by C1s into C4a and C4b, exposing sulfhydryl group that rapidly forms an amide or ester bond with nearby molecules containing hydroxyl or amino groups and forms C4d. C4d makes a covalent bond with tissue and stays at the site of complement activation for a long time, in comparison to other complement pieces. Identification of C4d in renal biopsy has been introduced first under diagnostic criteria of AMR in the year 2003.
Pathogenesis of complement 4d (Complement System and Complement 4d)
Complement is part of innate and adaptive immunity. It is an effector element of adaptive humoral immunity. Their activation depends on three different pathways; (classical, alternative, and lectin). This results in C4d which forms a covalent bond to injured tissue for a long time, and membrane attack complex (membrane-bound C5b—9) which destroys the cells.
Clinical relevance of Complement 4d:
1- Conditions associated with C4d positivity:
a- Acute AMR
b- Chronic AMR
c- A, B, O blood group ABO incompatible graft
d- Accommodation.
2- C4d negative states:
a- T-cell mediated rejection (TCMR)
b- A technical error like a type of fixatives, IF, versus IHC
c- Fc receptor (FCR) on NK cells (FcRIIA0 mediated rejection
d- Non-fixing antibodies (AB that cannot fix complement)
e- Complement independent pathways of endothelial activation
f- C4d deposition is very low to be identified by IF/IHC
g- Alloantibodies can directly damage the endothelium by interacting with MHC antigen
h- An increased expression of endothelial transcripts causing endothelial injury.
AMR occurs due to alloantibodies, it is also seen with high renal endothelial transcript expression and is an indicator of active antibody-mediated allograft damage and poor graft outcome.
Antibody-mediated rejection:
Responsible for 20—30% of graft loss. It has 3 types; hyperacute, acute, and chronic form. Presensitization is a major risk of hyperacute and acute AMR. Acute AMR is mostly occurring due to class I and II antibodies whereas, chronic AMR is largely due to Class II.
Other antigens which are responsible for AMR include MICA and ABO blood group antigen.
The diagnosis of AMR depends on the collaboration of histological findings (glomerulits, peritubular capillaritis, fibrinoid necrosis of arteries, and acute tubular injury), presence of DSA and C4d deposition in peritubular capillaries (PTCs).
1- Hyperacute AMR occurs within minutes to 24 hrs after transplantation, it is mainly due to preformed DSAs in high titres. The graft appears swollen, cyanotic and flaccid, microscopically, with arteritis, interstitial oedema and extensive cortical necrosis.
2- Acute AMR, C4d is deposited in PTC and glomerular capillaries.
3- Chronic AMR is characterized by TG which is defined by glomerular basement membrane duplication or splitting and mesangial expansion of glomeruli, tubular atrophy and interstitial fibrosis, and fibrous intimal thickening in arteries. Sometimes, electron microscopy also showed peritubular capillary basement membrane multilayering.
The mechanism of AMR includes:
1- Direct effects of DSA
2- 2- complement fixation
3- Cellular FCRs
Diagnosis of acute AMR depends on detection of DSA in plasma, C4d staining in PTC, and structural graft injury in presence of graft dysfunction.
Transplant glomerulopathy: TG
It is a form of chronic AMR, caused by humoral immunity. Morphologically TG consists of diffuse multilayering or duplication of the GBM. TG does not have discrete de novo or repetitive glomerular lesion, or evidence of thrombotic microangiopathies (TMA). Double contour of GBM occurs due to subendothelial accumulation of fluff (material and mesangial cells). In TG IF and IHC show, C4d deposition along the GBM presented in a minority of cases.
Transplant Glomerulopathy also associated with multilayering of peritubular capillarie
TG features consist of cytoplasmic vacuolation, broading of subendothelial space, with the existence of electron-dense floccular material. Interstitial fibrosis and tubular atrophy. New-onset arterial intima fibrosis, with leukocytic infiltration in intima and absence of internal elastic lamella, favours chronic rejection with TG.
TG was connected with the diagnosis of AMR, in the existence of either peritubular capillaritis or glomerulitis or both as well as with C4d expulsion in PTCs.
Complement 4d detection in renal allograft biopsy:
IHC and IF are techniques used to identify C4d in allograft biopsies.
IF
IHC
Applied for the Frozen section
Applied for paraffin-embedded tissue
C4d is described as widespread, strong linear circumferential PTC staining excluding the area of scar or necrosis
C4d is crisp, linear, continuous, diffuse, and lying around in PTC wall
Its strength is slighter and changeable
Have a fine granular pattern
Scoring is significant if C4d2 or C4d3
C4d > 0
C4d staining in 10% or more
C4d staining in 1% or more of PTC
More specific
Requires extra tissue
Used if biopsy small
More sensitive
Lower sensibility
Frozen section facility
Non-specific ground staining
Costly
More time consuming
Needs external control
DSA are most specifically associated with C4d staining in the PTC.
current Status of complement 4d
C4d is not a sensitive marker of AMR. MVI correlated with dispersing C4d positive cases in contrast to focal C4d positive cases. Currently, C4d has become an essential investigation for AMR according to Banff update 2017. In this update, both C4d and validated transcripts/classifiers/molecular markers can serve as potential alternatives and complements to DSAs in the diagnosis of ABMR.
complement 4d negative antibody‑mediated rejection
The morphology of C4d‑positive and C4d‑negative AMR has similar features:
1- glomerulitis and peritubular capillaritis.
2- frequent TCMR.
3- both may occur early or late post-transplantation.
C4d negative AMR has higher intrarenal endothelial gene expression, alloantibodies expression, and poor graft outcome usually occurs after 1-year post-transplantation and is associated with acute on chronic AMR.
Another type of AMR DSA negative AMR, in which DSA cannot be detected, moderate MVI with (g+ptc) scores ≥ 2 as per Banff 2013 with or without C4d positivity.
In ABO-incompatible graft transplantation, there is C4d deposition in PTCs in the absence of active rejection with or without DSApositivity, this process call accommodation.
Conclusion:
C4d appears to be a less sensitive marker than initially thought. It is a diagnostic as well as a prognostic marker for allograft.
PROS of C4d:
1- Provoked an enormous insight into the diagnosis of rejection.
2- It is a core diagnostic tool for AMR
3- C4d deposition pattern used in research in different clinical settings.
CONS of C4d
1- Difficulties in interpreting focal staining pattern
2- Low sensitivity of it in late renal allograft biopsies
3- Its lack of utility as a marker for antibody-mediated injury in ABO-incompatible allografts.
Despite all pitfalls, C4d is an excellent marker for AMR
Immune florescence
1- Applied for Frozen section
2- C4d described as widespread, strong linear circumferential PTC staining excluding area of scar or necrosis
3- Scoring is significant if C4d2 or C4d3
4- C4d staining in 10% or more
5- Requires extra tissue
6- More sensitive
require Frozen section facility
IHC
1- Applied for paraffin-embedded tissue
2- C4d is crisp, linear, continuous, diffuse, and lying around in PTC wall
3- Its strength is slighter and changeable
4- Have a fine granular pattern
5- C4d > 0 considered significant
6- C4d staining in 1% or more of PTC
7- More specific
8- Used if biopsy small
9- Lower sensibility
10- has non- specific ground staining
11- Costly
12- More time consuming
13- Needs external control
the system does not take tabulation therefore there is overlap between IF and IHC characters from IF until the external control are mixed
-The Banff criteria divided the rejection broadly into antibody‑mediated rejection (AMR), and cell‑mediated rejection. AMR is a predominant cause of allograft failure.
-Complement 4d (C4d) (that is part of component C4) is a fragment of the classical complement pathway, which is activated by immune complexes. C4 is cleaved by C1s into C4a and C4b, exposing a sulfhydryl group. The C4b forms C4d. C4d stays at the site of complement activation for a longer time, in comparison to other complement pieces.
-The diagnosis of AMR is improved by identification of the complement pieces
C4d in renal biopsy.
– Donor‑specific antibodies, are now considered by pathologists and clinicians as a significant cause of early and late graft dysfunction and failure.
– Clinical Relevance of Complement 4d
In the conditions where complement 4d is positive Following conditions are showing C4d positivity:
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A ,B, O blood group ABO-incompatible grafts
d. Accommodation.
The state where complement 4d is negative
The following conditions are showing C4d negativity:
a. T‑cell‑mediated rejection (TCMR)
b. A technical error like a type of fixative s , immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial
activation
f. C4d deposition is very low in quantity for the
identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact
h. Increased expression of endothelial transcripts causing endothelium injury.
-The C4d‑negative AMR occurs due to alloantibodies to MHC molecules on endothelial cells, elicit strong responses that include proliferation and activation of intracellular signaling pathways, which leads to endothelium injury.
-The C4d‑negative AMR is also seen with high renal endothelial transcript expression in patients with alloantibody and is an indicator of active antibody-mediated allograft damage and poor graft outcome.
-Antibody‑Mediated Rejection(AMR) accounts for up to 30% of all posttransplant
rejection events and 20%–30% graft loss at 1 year in untreated
cases.
-AMR can occur in three forms, namely hyperacute, acute, and chronic rejection. The presensitization (blood transfusion, pregnancy, and prior transplant) is a primary risk factor for hyperacute and acute rejection.Acute AMR is most commonly occurs due to expose of MHC Class I and II HLA antigens. Whereas, chronic AMR is largely associated with Class II DSA.
-Ther antigens that are responsible for AMR included MHC antigen (polymorphic) MICA (MHC Class I‑related chain A) and ABO blood group antigens.
-Hyperacute AMR is defined as rejection that occurs within minutes or 24 h after transplantation, mostly due to preexisting DSA present in high titers present in a patient. Grossly, the graft rapidly becomes cyanotic and flaccid. In acute AMR, C4d is deposited in PTC and glomerular capillaries. In chronic AMR, histomorphology characterized by transplant glomerulopathy (TG) in renal
biopsies.
-Transplant Glomerulopathy(TG ) is a variant of chronic AMR and is characterized by deposition of C4d. Morphologically, TG consists of diffuse multilayering or duplication of the glomerular basement membrane (GBM).
In TG double counter of GBM occurs due to the accumulation of fluff such as material
-The circulating DSA is most specifically associated with C4d deposition in PTC and its interactivity with endothelial cells in the graft.
– C4d has become an essential investigation for AMR according to Banff update 2017.
-The morphology of
C4d‑positive and C4d‑negative AMR having following similar
features:
(1) varying degrees of glomerulitis and peritubular Capillaritis.
(2) frequent TCMR.
(3) both may occur early or late posttransplantation.
-C4d‑negative AMR morphologically have higher intrarenal endothelial gene
expression, alloantibodies expression, poor graft outcomes, usually occur after 1‑year posttransplantation, and associated acute on chronic AMR.
-The accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity, mostly seen in ABO‑incompatible graft transplantation.
-C4d detection by IHC is more specific and C4d detection by IF is more sensitive. The IHC method for C4d detection is feasible in formalin‑fixed, paraffin‑embedded tissue, it is used when frozen sections facility is not available, IHC also useful when
small biopsy tissue or tissue not available for frozen sections.
-The disadvantage of C4d staining by IHC are lower sensibility, nonspecific background staining, more costly and more time consuming, and it needs external controls.
– IF method for C4d requires extra tissue and frozen sections facility.
The knowledge about role of C4 d in diagnosing AMR has evolved over the years . This article again has explored the relevance of C4d in this regard.
Complement 4d (C4d) (that is part of component C4) is a fragment of the classical complement pathway, which is activated by immune complexes. Complement is an important component of innate and adaptive immunity in our body. Complement is the leading effector constituent of adaptive humoral immunity.
The conditions where complement 4d is positive
Following conditions are showing C4d positivity
a. Acute AMR
b. Chronic antibody‐mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation.
The state where complement 4d is negative
The following conditions are showing C4d negativity
a. T‐cell‐mediated rejection (TCMR)
b. A technical error like a type of fixatives,
immunofluorescence (IF) versus immunohistochemistry
(IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated
rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial
activation
f. C4d deposition is a very low in quantity for the
identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact
with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing
endothelium injury.
Different techniques for detecting C4d are IF and IHC.
IF technique applied to frozen sections whereas IHC technique used to paraffin‐embedded tissue used routinely in various laboratories. In acute and chronic AMR‐positive C4d stain with IF technique is described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to a consensus at 2003 Banff Conference.[6] In IHC, C4d staining is crisp, linear, continuous, diffuse, and lying around in the PTC wall, while the strength typically is slighter and changeable, but it may have a finely granular pattern in high power.[40] The scoring of C4d is significant by IF on frozen section ie C4d2 or C4d3 and by IHC C4d >0 on paraffin sections.
According to various studies worldwide, C4d appears to be a less sensitive marker than initially thought however it should always be interpreted in a given clinical scenario
Introduction
C4d is one of diagnostic criteria of antibody mediated rejection.
It is a fragment of the classical complement pathway, makes covalent bond with the tissue and remain at site of complement activation for long time.
Complement is part of adaptive humoral immunity.
It is activated by 3 pathways, the classical pathway, the lectin pathway and may be activated by CRP.
Clinical relevance of C4d
Conditions were C4d is positive:
Acute antibody mediated rejection
Chronic antibody mediated rejection
ABO blood group incompatible grafts
Accommodation
Conditions with C4d negative:
T-cell mediated rejection
Fc receptor on NK cells mediated rejection
Technical error
Antibodies unable to fix complement
Complement independent pathway of endothelial activationC4d is low in quantity for Identification limits
Increased expression of endothelial transcripts causing endothelial injury.
Antibody mediated rejection (AMR):
In acute AMR, C4d is deposited in peritubular and glomerular capillaries.
Chronic AMR is characterized by transplant glomerulopathy in renal biopsy
The effector mechanism of AMR include direct effect of antibodies to MHC, complement fixation and cellular FCRs.
Diagnosis of AMR requires detection of DSA in serum, deposition of C4d in PTC and histological evidence of AMR
Absence of C4d deposition in PTC and DSA in serum decrease the probability of AMR.
Transplant glomerulopathy (TG):
Variant of chronic AMR, characterized by deposition of C4d.
Consists of diffuse multilayering or duplication of glomerular basement membrane which is best seen by periodic acid Schiff stain and silver stain.
TG also associated multilayering of peritubular capillaries:
Interstitial fibrosis and tubular atrophy exist with TG
New arterial intimal fibrosis with leukocytic infiltration in intima and absence of internal elastic lamellae favors chronic rejection with TG.
C4d detection in allograft biopsies:
Immunohistochemistry (IHC) and immunofixation (IF) are used in identification of C4d.
IF is applied to frozen sections, C4d stain is described as widespread, strong linear circumferential PTC staining, C4d2 or C4d3 is significant score
while IHC is applied to paraffin embedded tissue used routinely in various labs, C4d staining is crisp, linear and diffuse deposition in PTC wall, C4d>0 is significant score.
IF is more sensitive while IHC is more specific, used when frozen section is not available.
Current status of C4d:
C4d is not very sensitive marker for AMR.
Interobserver variation of IHC staining is improved with the binary scoring system.
According to Banff 217, C4d is an essential investigation for AMR
C4d and validated transcripts are potential alternatives to DSA in diagnosis of AMR.
C4d negative AMR:
It was included in Banff 2013 classification, Both C4d positive and C4d negative AMR have same different degrees of glomerulitis and peritubular capillaritis, frequent T cell mediated rejection and may occur early or late post transplant.
C4d negative AMR have higher antibody expression, poor graft outcome, usually occur after one year, may be acute or chronic and have increased intrarenal endothelial gene expression.
Accommodation is defined as C4d deposition in PTC in absence of active rejection with or without DSA seen in ABO incompatible transplant.
C4d staining is diagnostic and prognostic marker
Pros:
Used in diagnosis of AMR
Used in research in deposition patterns of C4d in different clinical settings.
Cons:
Difficulties in interpreting focal staining patterns
low sensitivity
Can’t be used as marker for AMR in ABO incompatible transplant
Introduction:
C4d is a degradation product of the classical complement pathway, which is activated by antigen antibody complexes. These alloantigens can be HLA Class 1&11, Non HLA and ABO antigens located at vascular endothelium.
C4D in the allograft biopsy facilitating diagnosis of ABMRT
Pathogenesis of C4d:
C4 is cleaved by C1s into C4a and C4b, exposing a sulfhydryl group. The C4b forms C4d which stays at the site of complement activation for a longer time, in comparison to other complement pieces.
Conditions associated with C4D positive .:
C4d Negative Causes:
Antibody Mediated Rejection (AMR):
The AMR is very harmful to the graft especially in more sensitized patients.
It accounts for up to 30% of all posttransplant rejection events and 20-30% graft loss at 1 year in untreated cases.
AMR can occur as hyperacute, acute, and chronic rejection.
Complement 4d Detection in Renal Allograft Biopsies
C4D is detected by two techniques: IHC and IF in allograft biopsies.
IF technique for frozen sections and the IHC technique used to paraffin‑sections
C4D is positive when it is C4d2 or C4d3 by IF or C4d >0 by IHC
IHC is more specific and C4d detection but IF is more sensitive.
As per Banff updates AMR is diagnosed by
•AMR is one of the major causes of graft loss
It occurs due to allo-antibodies that interact with donor antigen initiating immune responses.
It may acure as hyperacute within minutes to 24 hours, acute or chronic based on the degree of sensitization and Mismatchs between donor and recipient.
•Acute AMR most commonly occurs due to exposure to MHC Class I and II HLA antigens. chronic AMR is associated with Class II DSA.
There are also other antigens responsible for AMR includes MICA (MHC Class I‑related chain A) and ABO blood group antigens.
Complement system activationcan occure by three ways ,and is considered as the major factor of allograft injury in AMR.
C4d is a degradation product of the classical complement pathway, which is activated by antigen antibody complexes.
●conditions where complement 4d is positive
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO incompatible grafts d. Accommodation.
According Banff diagnostic criteria, C4d staining in PTCs (at least 10% PTC positive is considered as significant) by IHC on paraffin sections, Banff score C4d > 0 is significant.
by IFs on frozen sections Banff scores C4d2 or C4d3 is considered significant.
Thus, it is indicated that C4d detection by IHC more specific and C4d detection by IF is more sensitive.
●The state where complement 4d is negative: a. T‑cell‑mediated rejection (TCMR)
b. technical error .
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can directly cause endothelium injury
h. Increased expression of endothelial transcripts causing endothelium injury.
*Mixed cellular and humoral rejection are very hard to examine
AMR was considered the main cause of graft damage if there is positive IHC for C4d in PTC + high levels of DSA in the serum than cellular rejection.
*diagnostic features of AMR:
-presence of DSA andC4d deposition in peritubular capillaries (PTCs) in biopsy
– with other Microscopic findings of AMR; glomerulitis, peritubular capillaritis, fibrinoid necrosis of arteries, and acute tubular injury.
*chronic AMR, histomorphology characterized by transplant glomerulopathy (TG) in renal biopsies.
TG is a variant of chronic AMR and characterized by deposition of C4d. It proposed that TG may be one manifestation of humoral rejection of graft injury.
*The TG appears as:
capillary basement membrane duplication and mesangial expansion of glomeruli,
tubular atrophy and interstitial fibrosis,
fibrous intimal thickening in arteries. Sometimes, electron microscopy also showed peritubular capillary basement membrane multilayering
*The effector mechanisms of AMR includes
(1) Direct effects of antibody to MHC.
(2) Complement fixation.
(3) Cellular FCRs
•Current Status of complement 4d
C4d is not very sensitive marker of AMR, as it originally thought. Histologically, moderate to marked microvascular inflammation (MVI) correlated with dispersing C4d positive cases in contrast to focal C4d‑positive cases.
C4d has become an essential investigation for AMR according to Banff update 2017.
In this update, both C4d and validated transcripts/classifiers/molecular marker can
serve as potential alternatives and complements to DSAs in the diagnosis of ABMR.
•C4d negative antibody‑mediated rejection:
The C4d‑negative AMR was included in Banff 2013 classification.
The C4d‑negative AMR occur due to alloantibodies to MHC molecules on endothelial cells, elicit strong responses that include proliferation and activation of intracellular signaling pathways, which leads to endothelium injury.
The C4d‑negative AMR also seen with high renal endothelial transcript expression in patients with alloantibody and is an indicator of active antibody‑mediated allograft damage and poor graft outcome.
The morphology of C4d‑positive and C4d‑negative AMR having following similar features: (1) varying degrees of glomerulitis and peritubular capillaritis,
(2) frequent TCMR.
(3) both may occur early or late posttransplantation.
C4d‑negative AMR morphologically have higher intrarenal endothelial gene expression, alloantibodies expression, poor graft outcomes, usually occur after 1‑year posttransplantation, and associated acute on chronic AMR.
•conclusions
*The C4d‑positive staining in PTC is a diagnostic marker as well as a prognostic marker for allograft.
*C4d is less sensitive marker , some cons are :1)difficulties of interpreting focal staining patterns
(2) relatively low sensitivity of C4d as a marker for AMR in late renal allograft biopsies.
(3) its lack of utility in biopsies of ABO‑incompatible allografts.
Therefore, the detection of AMR should be best reported based on morphological features such as tubulointerstitial, vascular, and glomerular histological changes, with a piece of legislation to the presence or absence of C4d
the Banff criteria divided the rejection into antibody‑mediated rejection (AMR), and cell‑mediated rejection.
AMR is a predominant cause of allograft failure caused by antibodies against human leukocyte antigen (HLA) antigens defined as donor‑specific antibodies(DSA) which can cause early and late graft dysfunction.
(C4d) is part of component C4 is a part of the complement pathway. C4 is cleaved by C1s into C4a and C4b, exposing a sulfhydryl group which rapidly makes covalent bond with the tissue and stays at the site of complement activation for a longer time and can be used as a fingerprint for ABMR.
Pathogenesis
Complement 4d (Complement System and Complement 4d) Complement is an important component of innate and adaptive immunity in our body. Complement is the leading effector constituent of adaptive humoral immunity. There are three different pathways of complement activation and their activator.
· In the classical pathway of complement kick over of C1 (which is composed of C1q, C1r, and C1s) is begun by
1. an interplay of C1q with immunoglobulin bound to epitopes on the graft endothelium.
2. C1s(s>split) splits C4 into C4a and C4b and reacts by exposing a sulfhydryl group. The ester or amide bond forms with the interaction between the sulfhydryl group of C4b and nearby molecules containing hydroxyl or amino groups in the tissue.
3. C3 convertase (C4bC2a) of classical pathway is formed by a combination C4b with enzymatically loose pieces of C2a. C4bC2a (C3 convertase) splits C3 into C3a and C3b. C3b has an active sulfhydryl group that is dative bound and settles in the immediate locality and forms C5 convertase (C4bC2aC3b).
4. C5 convertase split C5 into C5a and C5b. Further, membrane attack complex (membrane‑bound C5b‑9) formed by the addition of C5b component of C5 convertase, that destroys cells.
· The lectin pathway of complement activates by mannan‑binding lectin (MBL) and H‑ficolin or L‑ficolin.
1. The MBL attaches to the suitable carbohydrate on apoptotic cells or pathogens)>not Ag X Ab.
2. MBL, H‑ficolin, and is L‑ficolin are homologous to C1q and fibrinogen(C1Q not involved in MLP), which are also activated C4 by their associated serine proteases, mannose-associated serine protease 1 (MASP-1 and MASP-2) (analogs to C1r and C1s).
· The C‑reactive protein (CRP) also activates the complement (C4) pathway. The CRP binds to carbohydrate and choline phosphate along with C1q (INVOLVED).
The following conditions are showing C4d positivity:
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO-incompatible grafts
d. Accommodation.
The following conditions are showing C4d negativity:
1. T‑cell‑mediated rejection (TCMR)
2. A technical error like a type of fixatives, immunofluorescence (IF) versus immunohistochemistry (IHC)
3. C4d deposition is a very low in quantity for the identification limits of IF/IHC
4. complement independent mechanism include Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection and endothelial activation as alloantibodies can direct endothelium injury to interact with major histocompatibility (MHC) Increased expression of endothelial transcripts causing endothelium injury.
5- Antibodies unable to fix the complement (IgG 2,4) complex
The C4d‑negative AMR is also seen with high renal endothelial transcript expression in patients with alloantibody and is an indicator of active antibody-mediated allograft damage and poor graft outcome.
Antibody‑Mediated Rejection
The AMR harms allograft kidney, chiefly in more sensitized patients. AMR accounts for up to 30% of all posttransplant rejection events and 20%–30% graft loss at 1 year in untreated cases.
AMR can occur in three forms, namely hyperacute, acute, and chronic rejection. The presensitization (blood transfusion, pregnancy, and prior transplant) is a primary risk factor for hyperacute and acute rejection.
Complement 4d Detection in Renal Allograft Biopsies
Two techniques that are mostly used in the detection of C4d include IHC and IF in allograft biopsies. IF technique is applied to frozen sections whereas the IHC technique used to paraffin‑sections
IF technique there is linear deposition along with PTC”
In IHC, C4d staining is linear, and diffuses along the PTC wall but it may have a finely granular pattern in high power.
The scoring of C4d is significant by IF on frozen sections ie C4d2 or C4d3 and by IHC C4d >0 on paraffin sections.
IHC is more specific and C4d detection by IF is more sensitive.
The disadvantage of C4d staining by IHC are lower sensibility, nonspecific background staining, more costly and more time consuming, and it needs external controls.
The C4d as a biomarker has the following
PROS :
(1) specific in the diagnosis of ABMR
(2) deposition patterns of C4d and sites of deposition can be used in DD
CONS :
(1) focal staining patterns make it difficult in interpretation
(2) low sensitivity
(3) not useful in biopsies of ABO‑incompatible allografts.
as per Banff updates
AMR diagnosis based on the presence of all 3 criteria
so Despite all pitfalls, C4d is an excellent marker for AMR
aftre renal transplantation, in case of the presence of antibodies, immune complexes form and react with real endothelium resulting in rejection and graft loss. this is hyperacute if happens within minutes up to 24 hours. other scenarios are acute and chronic rejection. hyperacute is usually due to preformed antibodies. acute AMBR can be due to either class I or II while chronic rejection is usually due to class II.
c4d is a by-product of the classical pathway. c4d is usually stained positive in case of acute AMBR, chronic ABMR, ABO-incompatible grafts, and in accommodation (mostly seen in ABO-incompatible transplants). accommodation is defined as C4d deposition in PTC in the absence of active rejection with or without DSA. C4d may be negative despite rejection. this may be cellular rejectşon or antibody-mediated but due to a third party. some antibodies are non-complement fixing. low levels of complement (below the threshold of detection). alloantibodies may lead to c4d -ve ABMR.
In acute AMBR c4d is deposited in PTC (peritubular capillaries) and glomerular capillaries. chronic ABMR is characterized by (TG) transplant glomerulopathy. TG is chracterşsed by duplication and multilayering of BM.
later both mixed cellular and ABMR may coexist but dşfferentiaition is hard because of the coexistence of both acute and chronic levels. ABMR is usually discarded in case of undetected DSAs and c4d negativity.
TG is a variant of chronic ABMR.
complement 4d is detected by either IF or IHC. IF is detected in frozen sections while IHC is used for paraffin-embedded tissue.
c4d negative ABMR included first in Banff 2013 has the same morphology as positive ABMR (varying degrees of glomerulitis and peritubular capillaritis, frequent TCMR and may occur early or late. however, c4d negative ABMR may have poor outcomes.
-Summary:
Banff criteria classify rejection into AMR and TCMR.
In AMR with +ve DSAs immune complexes activate the classical complement pathway giving rise to C4d.
Rejection with –ve C4d negative stain in the presence of AMR can be due to technical error , Fc receptor on NK cell mediated rejection, non-complement fixing antibodies, low quantity of C4d deposits ,non-HLA antibodies, and in antibodies causing direct endothelial injury.
Past history of sensitization is associated with hyper-acute and acute rejection. Acute AMR is associated with both class I and II DSA while chronic AMR is mainly associated with class II DSA.
Histopathology of AMR include glomerulits, peritubular capillaritis, fibrinoid necrosis and acute tubular injury and C4d deposition in peritubular capillaries.
C4d deposition is less in TG.
It has been shown that C4d is not a very sensitive marker for AMR. In Banff 2017 update, for a diagnosis of AMR, in addition to DSA and biopsy findings , either C4d or molecular markers can be used.
RELEVANCE OF C4d STAINING IN RENAL ALLOGRAFT BIOPSY
SUMMARY
The given article looks into the formation of C4d and its role in pathogenesis of AMR and its identification via biopsy.
C4d is a part of the complement pathway C4 and is formed by cleavage of C4 into the C4d and C4d to form C4d. C4d covalently bonds with the graft tissue and AMR can be diagnosed through testing for C4d presence via renal graft biopsy.
C4d positive is seen in following conditions
C4d negative AMR is seen in following conditions
AMR affects the allograft mainly in previously sensitized patients. It can occur as hyper acute, acute or chronic rejection. Hyperacute and acute rejection is seen in presensitized patients.
Transplant glomerulopathy is characterized by C4d deposition and is a variant of chronic AMR. Diffuse multilayering or duplication of the glomerular basement membrane is a distinct feature of this type of rejection. This duplication can be recognized with periodic acid Schiff staining methods.
C4d is a good tool in diagnosing AMR, both acute and chronic. However, it is not the best marker available and is less sensitive than it is thought to be.Discerning differences in focal patterns, low sensitivity in late biopsies as well as lack of diagnostic power in terms of ABO incompatible transplants all serve as points against C4d. However it is overall an excellent marker for AMR which should be included in making the ultimate diagnosis of rejection.
The Relevance of Complement C4d Staining in Renal Allograft Biopsies.
Introduction:
C4d is a degradation product of the classical complement pathway, which is activated by antigen antibody complexes. Complement system activation is the major contributor of allograft injury in antibody mediated rejection which can occur by three major pathways: classical, lectin, and alternative. These alloantigens can be HLA Class 1&11, Non HLA and ABO antigens located at vascular endothelium. The diagnosis ABMR is facilitated by incorporation C4d in the allograft biopsy. This review aims to evaluate pathogenesis and current relevance of C4d in ABMR.
Pathogenesis of C4d:
Classical pathway is activated when the C1 complex (C1q, C1r and C1s serine proteasess), binds to the Fc region of complement-fixing antibodies attached to the antigen. Activation of C1r and C1s in turn cleaves C4 and C2 into larger (C4b, C2a) and smaller (C4a, C2b) fragments.. C4b is converted to C4d which is main hallmark of ABMR. C4d staining has an important role in diagnosis of Acute and chronic ABMR.
C4d positive associated condition:
C4d Negative Causes:
Antibody Mediated Reaction (AMR):
Transplant Glomerulopathy (TG):
Conclusion
Anti-HLA DSAs are essential in both early and late graft survival. C4d is a fragment of complement classic pathway. It remains at the site of complement activation for a long time. Diagnosis of C4d in kidney biopsy has provided better yield for diagnosis of AMR. The complement system is a crucial component of innate and adaptive immunity. The complement system has three different path way.
Activation of the classical pathway starts with the interplay of C1q with immunoglobulin bound to epitopes on the graft endothelium. C4 is split into C4a and C4b and then C4b is converted to C4d. Activation and continuation of complement cascade by formation of C3 convertase and C5 convertase ultimately leads to formation of MAC and cell lysis. The lectin pathway, which is activated by MBL, H-ficolin, and L-ficolin also activates C4. In addition, CRP activates the complement C4 pathway.
The conditions with positive C4d staining are acute AMR, chronic AMR, ABO incompatible transplantation (accommodation).
The conditions with negative C4d staining include TCMR, technical errors, FCRII mediated rejection, non-complement fixing antibodies, very low quantity of C4d deposition, direct endothelium injury by alloantibodies, increased expression of endothelial transcripts.
C4d negative AMR due to antibody against MHC molecules on endothelium that are correlated with high endothelial transcripts expression can lead to severe endothelium injury and poor graft outcomes.
AMR
AMR constitutes up to 30% of posttransplant rejection. There are three forms of AMR. Acute AMR is commonly associated with class I and class II HLA antigens and chronic AMR is mostly associated with class II HLA antigens. MICA and ABO antigens are also responsible for AMR. The peritubular C4d finding is one of pathological diagnostic features of AMR (besides peritubular capillaritis, glomerulitis, fibrinoid necrosis, and acute tubular injury). Hyper acute rejection that occurs during the first 24 hours after transplantation is characterized by arteritis, interstitial edema, and extensive cortical necrosis. In chronic AMR, transplant glomerulopathy with duplication or splitting of GBM, mesangial expansion and IFTA are characteristic features. C4d deposition may be seen along with the GBM and peritubular capillary multilayering in EM may also be seen in TG. Mixed cellular and humoral rejection is hard for diagnosis, and positive peritubular C4d staining in combination of high level of DSA is highly suggestive of AMR. Excessive NK cells have been seen in the biopsy of acute and chronic AMR. The frozen section IF technique and paraffin-embedded IHC technique are used for detection of C4d. C4d staining by IF described as widespread, linear circumferential PTC in cortex and medulla, sparing scar and necrosis, and by IHC described as script, linear, continuous PTC and finely granular in high power.
C4d is not a very sensitive marker of AMR. Moderate to marked MVI is associated with disseminated C4d positivity. in Banff 2017, C4d, transcripts and molecular markers are considered as an alternative or complement of DSA.
C4d negative AMR
Although C4d positive and C4d negative have some similar features such as glomerulitis and peritubular capillaritis, C4d negative AMR have higher intrarenal endothelial gene expression and is usually associated with late acute AMR and chronic AMR.
AMR DSA negative AMR is described in the absence of DSA and presence of moderate MVI. TMA is a complication of AMR. C4d staining in peritubular capillaries more than 10% by IF and more than 0 by IHC is considered significant. C4d detection by IHC is more specific and by IF is more sensitive. IHC is also useful for small tissue biopsy, but is related to higher cost, nonspecific background staining, and higher time consuming.
(C4d) is a fragment of the classical complement pathway, which is activated by immune complexes.
identification C4d in renal biopsy improved the diagnosis of AMR .
C4d positive in flowing condition
a. Acute AMR
b. Chronic antibodymediated rejection
c. ABO incompatible grafts
d. Accommodation.
C4d negative in flowing condition
a. TCMR
b. Technical error
c.minimal C4d deposition
d. Antibodies cannot be attached to complement
e. Complement independent pathways of endothelial
activation
f.injury by Alloantibodies
AntibodyMediated Rejection: accounts for about 30% of all post-transplant rejection events and 20%–30% graft loss at 1 year if untreated.There are three forms of AMR: hyperacute, acute, & chronic . Acute AMR associated with HLA Class I & II antigens while chronic AMR is associated with Class II DSA.
Diagnosis of acute AMR : Diagnosis by DSA level ,C4d deposition in PTC, clinically rising scr .
Transplant Glomerulopathy: duplication of the glomerular basement membrane (GBM) and interstitial fibrosis and atrophy.
Two techniques used for identification of C4d. C4d detection by IHC is more specific & C4d detection by IF is more sensitive .
PROS :
(a) provoked an enormous amount of insight in the diagnosis of allograft rejection;
(b)It is core diagnostic tool to identify AMR;
c) used for majority of researches into the deposition patterns of C4d in different clinical settings such as pregnancy, thrombotic complications
CONS:
a) difficulties of explanation focal staining patterns;
(b) low sensitivity of C4d as a marker for AMR in late renal allograft biopsies;
(c) its lack of utility as a marker for antibodymediated injury in biopsies of ABOincompatible allografts)
AMR IS apredominant cause of allograft loss. C4d is a component of C4 complement factor . its presence suggest activation of classical complement pathway . detection of C4d in biopsy improves AMR diagnosis .thas wy it is included in diagnosis of AMR in Banff 2003.
C4d makes covalent bond with tissue and stay in tissue for longer time .
Following conditions are showing C4d positivity:
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation.
The following conditions are showing C4d negativity:
a. T‑cell‑mediated rejection (TCMR)
b. A t e ch n ica l e r ror l i ke a t y p e of f i x at ive s , immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing endothelium injury.
AMR ACOUNT FOR 30% acute rejection .and 30% of graft loss at 1 year.
Acute AMR is associated with both class I and class II HLA Ag. Whereas chronic rejection largely associated with class I .
Hyperacute AMR is defined as rejection occurs within minutes
or 24 h after transplantation, mostly due to preexisting DSA
Acute AMR, C4d is deposited in PTC and glomerular capillaries. In Chronic AMR, histomorphology characterized by transplant glomerulopathy (TG) in renal biopsies.
The effector mechanisms of AMR
(1) Direct effects of antibody to MHC,
(2) Complement fixation,
(3) Cellular FCRs
Diagnosis of AMR based on three component
1- Presence of DSA
2- Feature of Ab –Ag reaction ( c4d …)
3- Hitopathological signs of rejection.
Some studies had shown by utilizing microarray investigation showed excessive NK cells in biopsies of patients with acute and chronic AMR .
Transplant Glomerulopathy
TG is a morphological change that is mostly seen in humoral rejection and related to immunological etiology. TG is a variant of chronic AMR and characterized by deposition of C4d. TG consists of diffuse multilayering or duplication of the glomerular basement membrane .
Glomerular duplication is best seen by silver and PAS stain .
TG was connected with the diagnosis of AMR, in the existence of either peritubular capillaritis or glomerulitis or both as well as with C4d expulsion in PTCs
C4d is detected by two technique
IF – is asensitive but not specific test ,it needs fresh frozen sample , and adequate tissue sample .
IHC – is more specific than IF but less sensitive , more costy, more time consuming ,can be done in paraffin embedded tissue .
The scoring of C4d is significant by IF on frozen section ie C4d2 or C4d3 and by IHC C4d >0 on paraffin sections.
Currently, C4d has become an essential investigation for AMR
according to Banff update 2017.
The morphology of C4d‑positive and C4d‑negative AMR having following similar features:
(1) varying degrees of glomerulitis and peritubular capillaritis,
(2) frequent TCMR,
(3) both may occur early or late posttransplantation.
However, C4d‑negative AMR morphologically have higher intrarenal endothelial gene expression, alloantibodies expression, poor graft outcomes, usually occur after 1‑year posttransplantation, and associated acute on chronic AMR.
There is another entity of AMR DSA negative AMR, which characterized negative DSA/lack of DSA detection, moderate MVI with (g + ptc) scores of ≥2) as per Banff 2013 with or without C4d positivity.
Accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity, mostly seen in ABO‑incompatible graft transplantation.
Graft dysfunction results from either TCMD or ABMR ( either C4d + ve or – ve )
In cases of C4d + ve ABMR , complement activation starts after binding of DSA against mismatched HLA with graft endothelium followed by activation of the4 classic complement pathway with endpridction of C4d particles that deposits in PTC and are characteristics for ABMR
C4d are positive in following situations
– ABMR
– C ABMR
– ABO incompitable transplant
– Graft accommodation
C4d are – ve in
– False negative due to technical error
– Fc receotors on NK cells mediated rejection
– Non complement fixing ab
– Non HLA Ab
Draw backs of C4d as a marker of rejection
– Interobserver variation in detecting and evaluating the degree of deposition
– Poor reducibilty
Two techniques are used for detection of C4d
in allograft biopsies
1- IF technique : requires frozen sections
– IHC technique requires paraffin‑embedded tissue and more routinely used than IF
For diagnosis of acute or chronic ABMR , C4d stain show
1- With IF , it appears as widespread linear and circumferential PTC staining in both cortex or medulla, excluding areas of scaring or necrotic areas . the significant C4d scoring by IF is >10%
2- In IHC : C4d staining gives linear, continuous, diffuse deposits
3- around in the PTC wall, the significant C4d scoring by IHC is C4d >0
Conclusion
Although C4d is not very sensitive marker of AMR and has several falicies , still its important diagnostic criterion of AMR according to Banff update 2017
The Relevance of complement C4d staining in renal allograft Biopsies
Complement is an important component of innate and adaptive immunity in our body,it activated and deposits in renal allograft after the attack of antibodies to the vascular endotheliam.
There’s some conditions showing C4d positive:
Acute AMR
Chronic AMR
ABO incompatible graft
Accommodation
Condition show negative C4d are:
T cell mediated rejection
Technical error
Fc receptor of NK mediated rejection
Ab unable to fix complement
Complement independent pathway of endothelial injury
Very low quantity of C4d to identification by IF or IHC
Allow antibodies directed to endothelium
Increase expression of endothelial transcript causing injury
So C4d detection in PTC has diagnostic and prognostic marker for allograft but had pros like it consider core diagnostic tool to identify AMR and used in many research but the cons were difficult to interpreting focal staining pattern and relatively low sensitive specially in the cases of ABO incompatible transplant
Complement 4d (C4d) (that is part of component C4) is a fragment of the classical complement pathway, which is activated by immune complexes. The diagnosis of AMR is improved by identification of the complement pieces C4d in renal biopsy, and it has been introduced first under diagnostic criteria of AMR in year 2003.
clinical relevance Of cOmplement 4d
The conditions where complement 4d is positive
a. Acute AMR
b. Chronic antibody‐mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation.
The state where complement 4d is negative
a. T‐cell‐mediated rejection (TCMR)
b. A technical error like a type of fixatives,
immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the
identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact
with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing endothelium injury.
The C4d‐negative AMR seen with high renal endothelial transcript expression in patients with alloantibody and is an indicator of active antibody‐mediated allograft damage and poor graft outcome.
The C4d‐negative AMR occur due to alloantibodies to MHC molecules on endothelial cells, elicit strong responses that include proliferation and activation of intracellular signaling pathways, which leads to endothelium
antibOdy‐mediated rejectiOn
AMR can occur in three forms, namely hyperacute, acute, and chronic rejection.
The presensitization (blood transfusion, pregnancy, and prior transplant) is a primary risk factor for hyperacute and acute rejection.[17] Acute AMR is most commonly occurs due to expose of MHC Class I and II HLA antigens.[17] Whereas, chronic AMR is largely associated with Class II DSA.
Besides, other antigens which are responsible for AMR included MHC antigen (polymorphic) MICA (MHC Class I‐related chain A) and ABO blood group antigens.
tranSplant glOmerulOpatHy
TG is a variant of chronic AMR and characterized by deposition of C4d.
Morphologically, TG consists of diffuse multilayering or duplication of the glomerular basement membrane (GBM).
cOmplement 4d detectiOn in renal allOgraft
biOpSieS
Two techniques are mostly used for identification of C4d in allograft biopsies of the kidney:
1. Indirect immunofluorescence (IF):
applied to frozen sections
In acute and chronic AMR‐positive C4d stain with IF technique is described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to a consensus at 2003 Banff Conference.
The scoring of C4d is significant by IF on frozen section ie C4d2 or C4d3
C4d is significant staining in 10% or more for frozen/frequency
C4d detection by IF is more sensitive.
Less expensive .
Rapid detection of C4d positivity.
IF method for C4d requires extra tissue and frozen sections facility.
Can not be stored for latter assessment.
Still the gold standard technique
2. Immunohistochemical (IHC):
IHC technique used to paraffin‐embedded tissue used routinely in various laboratories.
C4d staining is crisp, linear, continuous, diffuse, and lying around in the PTC wall, while the strength typically is slighter and changeable, but it may have a finely granular pattern in high power.
The scoring of C4d is significant by IHC C4d >0 on paraffin sections.
C4d detection by IHC more specific
The IHC method for C4d detection feasible in formalin‐fixed, paraffin‐embedded tissue, it is used when frozen sections facility not available, IHC also useful when small biopsy tissue or tissue not available for frozen sections.
The disadvantage of C4d staining by IHC are lower sensibility, nonspecific background staining, more costly and more time consuming, and it needs external controls.
IHC method Widely available.
Uses the same sample that is processed for LM .
cOmplement 4d negative antibOdy‐mediated
rejectiOn
The morphology of C4d‐positive and C4d‐negative AMR having following similar features: (1) varying degrees of glomerulitis and peritubular capillaritis, (2) frequent TCMR, (3) both may occur early or late posttransplantation.
There is another entity of AMR DSA negative AMR, which characterized negative DSA/lack of DSA detection, moderate MVI with (g + ptc) scores of ≥2) as per Banff 2013 with or without C4d positivity.
The accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity, mostly seen in ABO‐incompatible graft transplantation.
Thank You
Antibody-mediated rejection occurs more frequently in individuals who have been sensitized and accounts for 30 per cent of all transplant patients.
A history of exposure to pregnancy or transfusion, as well as a prior transplant, increases the risk of AMR, which can result in hyperacute rejection in most cases.
AMR may be classified into three types: hyperacute acute, acute, and chronic.
acute AMR related with MHC class I and class II exposure b persistent AMR linked with MHC class II exposure
AMR’s pathology is as follows:
glomerulitis, peritubular capillaries, fibrinoid necrosis of arteries, and acute tubular damage are all shown on a microscope in the presence of AMR. Gross enlargement of the kidney owing to interstitial oedema and bleeding is also seen on microscopy.
The presence of DSA and C4d deposits in the PTC is a diagnostic characteristic of AMR.
Causes of C4d positivity include:
. Acute Mycobacterial Respiratory Distress (AMR)
. AMR that is persistent
. Incompatibility with ABO
. T-cell-mediated rejection as a source of C4d negativity as an accommodation (TCMR)
. A technical mistake in the immunofluorescence (IF) or immunohistochemistry (IHC) procedure (IHC)
. Rejection is mediated by the Fc receptor (FCR) on natural killer cells (FcRIIA).
. Antibodies that are incapable of fixing the complement e. Endothelial activation mechanisms are not reliant on the complement.
The presence of C4d-negative AMR due to alloantibodies to MHC molecules on endothelial cells elicits significant reactions that include proliferation and activation of intracellular signalling pathways, which ultimately results in endothelium damage.
In addition, it is present in patients who have developed an alloantibody, and it is a sign of active antibody-mediated allograft destruction and poor graft fate.
AMR causes kidney damage in transplant recipients who are already sensitive. In situations when AMR is left untreated, it can account for up to 30% of all posttransplant rejection episodes, as well as 20%–30% of graft loss after one year after transplantation. AMR classifies rejection into three categories: hyperacute, acute, and chronic.
AMR occurs often in patients who have had a previous blood transfusion, are pregnant, or have had previous transplantation, among other things.
Acute AMR is most usually caused by exposure to MHC Class I and II HLA antigens, which is a common occurrence. Chronic AMR has been shown to be significantly related to Class II DSA.
Hyperacute AMR is characterized as rejection that develops within minutes or within 24 hours of transplantation and is mostly caused by preexisting DSA, among other factors. The graft becomes cyanotic and flaccid on a gross level very quickly. Arteritis, interstitial oedema, and widespread cortical necrosis define the histomorphology of this condition. C4d is accumulated in the PTC and glomerular capillaries of patients with acute AMR. Renal biopsies from patients with chronic AMR show histomorphology defined by transplant glomerulopathy (TG).
Positive staining for C4d in the PTC serves as both a diagnostic and a predictive indication for transplant failure.
Because of the unique properties of C4d, it has (a) provided a tremendous amount of insight into the diagnosis of allograft rejection, (b) served as a core diagnostic tool to identify AMR, and (c) been used for a vast amount of research into the deposition patterns of C4d in different clinical settings such as pregnancy, thrombotic complications, and the following CONS: (1) difficulties in interpreting focal staining patterns, (2) low sensitivity of C4d as a marker for AMR in late renal rejection.
Thank You
In renal transplant, the allograft is affected by many triggering agents such as innate and adaptive immune mechanisms, either mediated by macrophages and lymphocytes, or by soluble components antibodies and the complement system, which can ultimately lead to graft rejection. Antibody‑mediated rejection (AMR) is a predominant cause of allograft failure.
Pathogenesis of Complement 4d
Ag-Ab complexes activate the classical complement pathway with the binding of C4d to the endothelium of peritubular capillaries (PTCS) leading to ABMR
C4d positivity occurs in acute AMR, Chronic antibody‑mediated rejection, ABO incompatible grafts and Accommodation.
C4d negative AMR occurs in T‑cell‑mediated rejection (TCMR),due to technical error in type of fixatives(immunofluorescence (IF) versus immunohistochemistry (IHC), Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection, Antibodies unable to fix the complement, C4d deposition is a very low in quantity for the identification limits of IF/IHC , Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC), Increased expression of endothelial transcripts causing endothelium injury
Antibody‑Mediated Rejection: accounts for about 30% of all post-transplant rejection events and 20%–30% graft loss at 1 year if untreated.There are three forms of AMR: hyperacute, acute, & chronic . Acute AMR associated with HLA Class I & II antigens while chronic AMR is associated with Class II DSA.
Pathology of AMR: Gross-swollen kidneys(interstitial edema & hemorrhage). Microscopic – glomerulitis, PTCs, fibrinoid necrosis of arteries, & ATN. Other features include presence of DSA & C4d deposition PTCs.
Diagnosis of acute AMR : Diagnosis of acute AMR based on the identification of DSA in plasma, C4 complement component (C4d) in PTC, structural features of AMR, such as kidney injury and graft dysfunction
Transplant Glomerulopathy: TG may be one manifestation of humoral rejection of graft injury and consists of diffuse multilayering or duplication of the glomerular basement membrane (GBM) and interstitial fibrosis and atrophy.
Two techniques used for identification of C4d. C4d detection by IHC is more specific & C4d detection by IF is more sensitive .The scoring of C4d is significant by IF on frozen section i.e., C4d2 or C4d3 and by IHC C4d >0 on paraffin sections. C4d & validated transcripts & molecular marker can be potential alternatives & complements to DSAs in the diagnosis of ABMR.
The C4d as a biomarker has following PROS :
(1) provoked an enormous amount of insight in the diagnosis of allograft rejection;
(2) core diagnostic tool to identify AMR;
(3) used for vast amount of research into the deposition patterns of C4d in different clinical settings such as pregnancy, thrombotic complications
And has following CONS:
(1) difficulties of interpreting focal staining patterns;
(2) relatively low sensitivity of C4d as a marker for AMR in late renal allograft biopsies;
(3) its lack of utility as a marker for antibody‑mediated injury in biopsies of ABO‑incompatible allografts).
Thanks Batool
Post kidney transplant, the recognition of the transplanted organ as foreign is mediated by complex immunologic pathways, the Banff criteria divided the rejection in general terms into cellular (T-cell mediated) and humoral (B-cell mediated) pathways.
AMR is a predominant cause of allograft failure. ABMR is caused by antibodies directed against donor-specific human leukocyte antigen (HLA) molecules (DSA) which is considered as a remarkable cause of early and late graft dysfunction.
Complement 4d (C4d) (that is part of component C4) is a fragment of the classical complement pathway, which is activated by immune complexes for diagnosis and identification of the complement pieces C4d biopsy remains the gold standard and provides valuable insights into the pathogenesis of early and late allograft injury.
Complement is an important component of innate and adaptive immunity in our body. Complement is the leading effector constituent of adaptive humoral immunity. There are three different pathways of complement activation, immune complex activates the classical pathway when the c1q interplay with immunoglobulin bound to epitopes on the graft endothelium. C1s splits C4 into C4a and C4b and react with exposing a sulfhydryl group giving rise toc4d forming covalent bond with the tissue, C3 convertase (C4bC2a) of classical pathway is formed by combination C4b with enzymatically loose pieces of C2a. it splits C3 into C3a and C3b. C3b has an active sulfhydryl group and forms C5 convertase (C4bC2aC3b). C5 convertase split C5 into C5a and C5b. Further, membrane attack complex (membrane‑bound C5b‑9) formed by the addition of C5b component of C5 convertase, that destroys cells.
C4d positivity can be seen
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. ABO incompatible grafts
d. Accommodation
C4d negativity: can be seen
a. T‑cell‑mediated rejection (TCMR) b. A technical error like a type of fixatives, immunofluorescence (IF) versus immunohistochemistry (IHC) c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection d. Antibodies unable to fix the complement e. Complement independent pathways of endothelial activation f. C4d deposition is a very low in quantity for the identification limits of IF/IHC g. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC) h. Increased expression of endothelial transcripts causing endothelium injury.
The C4d‑negative AMR occur due to alloantibodies to MHC molecules on endothelial cells, elicit strong responses that include proliferation and activation of intracellular signaling pathways, which leads to endothelium injury. The C4d‑negative AMR also seen with high renal endothelial transcript expression in patients with alloantibody and is an indicator of active antibody-mediated allograft damage and poor graft outcome.
AMR can be hyper acute, acute, and chronic rejection. presensitization is a primary risk factor for hyper acute and acute rejection. Acute AMR is most commonly occurring due to expose of MHC Class I and II HLA antigens. Whereas, chronic AMR is largely associated with Class II DSA.
other antigens which has a role in AMR included MHC antigen eg: MICA and ABO blood group antigens.
Histological feature of ABMR – interstitial edema and hemorrhage.
Microscopic findings of AMR- glomerulitis, peritubular capillaritis, fibrinoid necrosis of arteries, and acute tubular injury, Other diagnostic features of AMR requires the presence of DSA and C4d deposition in peritubular capillaries (PTCs).
Hyper acute AMR is defined as rejection occurs within minutes or 24 h after transplantation, mostly due to preexisting DSA present in high titers present in a patient. Grossly, the graft rapidly becomes cyanotic and flaccid. Histomorphology characterized by arteritis, interstitial edema, and extensive cortical necrosis.
In acute AMR, C4d is deposited in PTC and glomerular capillaries.
In chronic AMR, histomorphology characterized by transplant glomerulopathy (TG) in renal biopsies.
TG is mostly seen in humoral rejection TG is a variant of chronic AMR and characterized by deposition of C4d. TG may be one manifestation of humoral rejection of graft injury. Morphologically, TG consists of diffuse multilayering or duplication of the glomerular basement membrane
The C4d‑negative AMR is included in Banff 2013 classification.The morphology of C4d‑positive and C4d‑negative AMR having following similar features: (1) varying degrees of glomerulitis and peritubular capillaritis, (2) frequent TCMR, (3) both may occur early or late posttransplantation.[ However, C4d‑negative AMR morphologically have higher intrarenal endothelial gene expression, alloantibodies expression, poor graft outcomes, usually occur after 1‑year posttransplantation, and associated acute on chronic AMR.
The accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity, mostly seen in ABO‑incompatible graft transplantation.[48] The TMA (thrombotic microangiopathy) is a complication of AMR in the absence of other causes characterized with fibrin thrombi, fragmented red blood cells, mesangiolysis, and muco‑intimal thickening and injury of small vessels.
III. The Relevance of Complement C4d Staining in Renal Allograft Biopsies
Please summarise this article
# The Banff criteria divided post transplantation rejection in antibody mediated rejection (AMR) and cell mediated rejection.
# AMR is a predominant cause of allograft failure and presence of the DSA consider a remarkable cause of early and late graft dysfunction.
# Complement (C4d) is a fragment of the classical complement pathway, which is activated by immune complexes.
# The diagnosis of AMR is improved by identification of the complement pieces C4d in renal biopsy.
# Complement is an important component of innate and adaptive immunity .
# There are three different pathways of complement activation by macrophages, lymphocytes, soluble components antibodies and the complement system
**classical complement pathway
Activation start with C1 ( C1q, C1r, and C1s)
C1q with immunoglobulin bound to epitopes on the graft endothelium. C1s splits C4 into C4a and C4b.Then C3 convertase (C4bC2a) splits C3 into C3a and C3b. C3b forms C5 convertase (C4bC2aC3b).
C5 convertase split C5 into C5a and C5b. followed by membrane attack complex (membrane bound C5b‑9)
**The lectin pathway of complement activates by Mannan binding lectin (MBL) , H‑ficolin or L‑ficolin.
The MBL attach to the suitable carbohydrate on apoptotic cells or pathogens.
** Alternative pathway
# The following conditions are showing C4d positivity:
* Acute AMR
* Chronic antibody mediated rejection
* ABO incompatible grafts
* Accommodation.
# Following conditions are showing C4d negativity:
*Tcell mediated rejection
* Technical error like a type of fixatives ,
immunofluorescence IF versus immunohistochemistry IHC
* Fc receptor on NK cells (FcRIIA) mediated
rejection
* Antibodies unable to fix the complement
* Complement independent pathways of endothelial activation
* C4d deposition is a very low in quantity for the identification limits of IF/IHC
* Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC)
* Increased expression of endothelial transcripts causing endothelium injury
# Antibody mediated rejection
# The AMR harms allograft kidney in sensitized patients, it accounts for up to 30% of all post transplant rejection and 20%–30% graft loss at 1 year in untreated cases.
# AMR Three types, hyper acute, acute, and chronic rejection.
The pre sensitization is a primary risk factor for hyper acute and acute rejection.
* Hyperacute AMR is defined as rejection occurs within minutes or 24 h after transplantation, mostly due to preexisting DSA present in high titres , the graft rapidly becomes cyanotic and flaccid, histomorphology characterized by arteritis, interstitial edema, and extensive
cortical necrosis.
*Acute AMR is most commonly associated with MHC class 1and II DSA
# Other antigen responsible for AMR included MICA (MHC Class I‑related chain A) and ABO blood group antigens
# Microscopic findings include glomerulitis, peritubular capillaritis, fibrinoid necrosis of arteries, and acute tubular injury also presence of DSA and C4d deposition in peritubular capillaries and glomerular capillaries leading to kidney injury and graft dysfunction
* Chronic AMR is largely associated with class II DSA.
There is glomerulopathy (TG) in renal biopsies, as capillary basement membrane duplication or splitting and mesangial expansion of glomeruli, tubular atrophy interstitial fibrosis, and fibrous intimal thickening in arteries and peritubular capillary basement membrane multilayering.
# The effector mechanisms of AMR:
* Direct effects of antibody to MHC
* Complement fixation
* Cellular FCRs
* Mixed cellular and humoral rejection are very hard occurs after months and years posttransplantation, and
biopsy show acute and chronic lesion.
# Transplant Glomerulopathy
TG is a morphological change that is mostly seen in humoral rejection and related to immunological etiology, is a variant of chronic AMR and characterized by deposition of C4d, diffuse multilayering or duplication of the glomerular basement membrane their lack distinct de novo or repetitive glomerular lesion or TMA
# Transplant Glomerulopathy Also Associated
Multilayering of Peritubular Capillarie
There is arterial intimal fibrosis of new onset, leukocytic
infiltration in intima indcating chronic rejection with TG.
TG was connected with the diagnosis of AMR of either peritubular capillaritis, glomerulitis or both as well as with C4d
expulsion in PTCs.
# Complement 4d Detection in Renal Allograft Biopsies C4d detected by using:
*IHC on paraffin embedded tissue; (crisp, linear, continuous, diffuse, and lying around in the PTC wall).
* IF applied to frozen sections (widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic area).
# Current Status of Complement 4d
Devadass et al. reported C4d is not very sensitive marker of
AMR
Banff update 2017 both C4d and validated transcripts/classifiers/molecular marker can serve as potential alternatives and complements to DSAs in the diagnosis of ABMR.
# Complement 4d Negative Antibody Mediated Rejection
The morphology of C4d positive and C4d negative AMR having following similar features:
* Varying degrees of glomerulitis and peritubular capillaritis *Frequent TCMR
* Both may occur early or late posttransplantation. C4d negative AMR morphologically have higher intrarenal endothelial gene, alloantibodies expression& poor graft outcomes occur after 1 year posttransplantation and associated acute on chronic AMR.
The accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity,
mostly seen in ABO incompatible graft transplantation.
# Conclusion
The C4d as a biomarker has following PROS
* Provoked an enormous amount of insight in the diagnosis of allograft rejection
* Core diagnostic tool to identify AMR
* Used for vast amount of research into the deposition patterns of C4d in different clinical settings
CONS of C4d
* Difficulties of interpreting focal staining
Patterns
*Relatively low sensitivity of C4d as a marker
for AMR in late renal allograft biopsies
*Its lack of utility as a marker for antibody mediated injury in biopsies of ABO incompatible allografts
Well done Dr Manal
Introduction:
Acute rejection is divided into Antibody-mediated rejection and cell-mediated rejection according to Banff’s classification C4d is a fragment of a classical complement pathway which helps in the diagnosis of antibody-mediated rejection by detection of C4d
Clinical importance of positive C4d :
Acute AMR
Chronic AMR
Incompatible ABO kidney transplantations
Accommodation
Antibody‑Mediated Rejection
may be hyperacute, acute, and chronic, responsible for 20-30 % of graft loss in first-year post-transplantation, and associated with DSA against Class I and Class II of HLA antigens or DSA to non-HLA antigens like (MICA and ABO blood antigens)
classified as :
Hyperacute rejection: with first 24 hours (cyanotic graft and cortical necrosis)
Acute : glomerulitis and peritubular capiliritis
Chronic: transplant glomerulopathy
Mixed Antibody and cellular: after months or years and biopsy may show acute or chronic affections
Transplant glomerulopathy:
pathology includes: subendothelial broadening, interstitial fibrosis, tubular atrophy, and intimal fibrosis of arteries
C4d detection in Biopsies :
IF C4d 2 or 3
IHC > 0
essential for diagnosis of acute ABM however decrease sensitivity in chronic ABM
C4d negative ABM rejection:
maybe associated with acute or chronic ABM rejection
with higher endothelial gene expression, Alloantibodies with poor graft survival
C4d positive with no ABM rejection
in ABO incompatible transplantation with accommodation
TMA
BK nephropathy
pregnancy
Conclusion
C4d as PROS :
1-has role in the detection of allograft rejection
2-diagnostic item in ABM rejection
3-detected in pregnancy and thrombotic events
C4d as CONS :
1-low sensitivity marker in ABM in late biopsies
2-difficult interpretations of C4d
3- decrease importance in ABO-incompatible allograft biopsies with antibody-mediated injury
Thank You
Summery:
Renal allograft is affected by many triggering agents such as innate and adaptive immunity which can lead to graft rejection. ABMR is the predominant cause of allograft failure, and DSA is a remarkable cause of early and late graft dysfunction. There is more development about C4d after inclusion in the diagnostic criteria of ABMR.
Clinical Relevance of C4d:
Positive C4d is seen in:
Negative C4d seen in:
ABMR:
accounts for 30% of all post transplant rejection events and 20-30% graft loss at 1 year in untreated cases.
ABMR can occur in 3 forms: hyper acute, acute, and chronic. Presensitization is the risk factor for hyperacute and acute rejection.
Pathological findings of ABMR is grossly swollen kidney due to odema and hemorrhage. Microscopic findings include glomerulitis, peritubular capillaritis, fibrinoid necrosis of arteries and acute tubular injury. Other diagnostic features are the presence of DSA and C4 d deposition in the peri tubular capillaries.
hyperacute rejection occurs within minutes to 24 hr post transplant due to preexisting DSA, acute rejection C4d is deposited in the ptc and glomerular capillaries while chronic ABMR is characterized by the presence of TG.
The effector mechanisms of ABMR are:
diagnosis of ABMR is based on the identification of DSA in plasma, C4d in ptc, and graft dysfunction.
TG:
mostly seen in humoral rejection related to immunological injury, characterized by C4d deposition. It consists of multilayering of GBM. IF and IHC show C4d deposition along the GBM presented in the minority of cases (6% in one series).
TG is also associated with multilayering of peri tubular capillaries:
TG was connected with the diagnosis of ABMR in the existence of either peri tubular capillaritis or glomerulitis or both as well as with C4d expulsion in PTCs.
C4d detection in allograft biopsies:
two techniques are used for identification of C4d, they are IHC and IF.
In ABMR, positive C4d staining with IF technique is described as widespread, strong linear circumferential ptc staining in cortex or medulla, excluding scar area according to Banff 2013.
In IHC C4d staining is crisp, linear, continuous, diffuse, and lying around in the ptc wall, it’s strength is slightler and changeable but it may have a fine granular pattern in high power.
The scoring of C4d is significant by IF on frozen section ( C4d2, C4d3) and by IHC C4d> 0 in paraffin sections.
The DSA is most specifically associated with C4d deposition in ptc and it’s interactivity with endothelial cells in the graft.
Current state of C4d:
studies showed that C4d is not very sensitive marker of rejection as it was thought originally. Currently C4d has become an essential investigation for ABMR according to Banff 2017. In this update, both C4d and validated transcripts/ classifiers/ molecular markers can serve as potential alternatives and complements to DSA in the diagnosis of ABMR.
C4d negative ABMR:
It was included in the Banff 2013 classification, has similar morphology to C4d positive rejection, but have higher intrarenal endothelial gene expression, alloantibody expression, poor graft outcomes, usually occur after 1 year post transplantation and associated with acute and chronic ABMR.
Thank You
Summary of The relevance of complements C4dstaining in renal allograft biopsy .
C4d is fragment of the classical complement pathway,
C4 is cleaved by C1s into C4a and C4b, exposing a sulfhydryl group. The active
sulfhydryl group of C4b rapidly forms an amide or ester bond with nearby molecules
containing hydroxyl or amino groups and forms C4d, C4d makes covalent bond with the
tissue. It stays at the site of complement activation for a longer time, in comparison to
other complement pieces. The diagnosis of AMR is improved by identification of the C4d.
The conditions where complement 4d is positive:
a. Acute AMR .
b. Chronic antibody‑mediated rejection.
c. A, B, O blood group ABO incompatible grafts .
d. Accommodation.
The state where complement 4d is negative The following conditions are showing C4d
negativity:
a. T‑cell‑mediated rejection (TCMR)
b. A technical error like a type of fixative IF versus IHC.
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement.
e. Complement independent pathways of endothelial activation.
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with major histocompatibility
complex (MHC).
h. Increased expression of endothelial transcripts causing endothelium injury.
Antibody‑Mediated Rejection:
The AMR harms allograft kidney, chiefly in more sensitized patients. (Transfusions,
pregnancy or previous transplantation)
AMR accounts for up to 30% of all posttransplant rejection.
and 20%–30% graft loss at 1 year in untreated cases.
AMR can occur in three forms:
Hyperacute.
acute. More to MHC class 1 and MHC class 11 antigens
chronic rejection, more due to MHC class 11 antigens
other antigens which are responsible for AMR included MHC antigen (polymorphic) MICA
(MHC Class I‑related chain A) and ABO blood group antigens
Complement 4d Detection in Renal Allograft Biopsies:
Two techniques of C4d IHC and IF in allograft biopsies of the kidney.
IF technique applied to frozen sections whereas IHC technique used to
paraffin‑embedded tissue used routinely in various laboratories.
In acute and chronic AMR‑positive C4d stain with IF technique is described as
“widespread, strong linear circumferential PTC staining in cortex or medulla, excluding
scar or necrotic areas,” according to a consensus at 2003 Banff Conference.
In IHC, C4d staining is crisp, linear, continuous, diffuse, and lying around in the PTC
wall, while the strength typically is slighter and changeable, but it may have a finely
granular pattern in high power.
The scoring of C4d is significant by IF on frozen section i.e., C4d2 or C4d3 and by IHC
C4d >0 on paraffin sections.
Current Status of Complement:
C4d is not very sensitive marker of AMR, as it originally thought
C4d is significant staining in 1% or more of the PTC for formalin/IHC‑IP, or 10% or more
for frozen/frequency IF.
Interobserver and interinstitutional reproducibility for C4d IHC staining in renal allografts
was poor but improved with a binary scoring system (positive/negative).
Currently, C4d has become an essential investigation for AMR according to Banff
update 2017.
Complement 4d Negative Antibody‑Mediated Rejection:
The C4d‑negative AMR was first demonstrated Feucht et al., and Edmonton group
The C4d‑negative AMR is included in Banff 2013 classification.
The morphology of C4d‑positive and C4d‑negative AMR having following similarfeatures:
(1) varying degrees of glomerulitis and peritubular capillaritis.
(2) frequent TCMR.
(3) both may occur early or late post transplantation.
However, C4d‑negative AMR morphologically have higher intrarenal endothelial gene
expression, alloantibodies expression, poor graft outcomes, usually occur after 1‑year
post transplantation, and associated acute on chronic AMR.
There is another entity of AMR DSA negative AMR, which characterized negative
DSA/lack of DSA detection, moderate MVI with (g + ptc) scores of ≥2) as per Banff 2013
with or without C4d positivity.
The accommodation is defined as C4d deposition in PTCs in the absence of active
rejection with or without DSA positivity, mostly seen in ABO‑incompatible graft
transplantation…
Thank You
Types of ABMR,
a-Hyperacute AMR,rarely seen now adays
b-Acute AMR,
c-Chronic AMR:
Complement 4d Detection in Renal Allograft Biopsies,
Clincal relevance of complement C4d
4- Accommodation.
1. Tcellmediated rejection (TCMR)
. 2 .A technical error like a type of fixatives, immunofluorescence (IF) versus immunohistochemistry (IHC)
3. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
4. Antibodies unable to fix the complement
5. Complement independent pathways of endothelial activation
6. C4d deposition is a very low in quantity for the identification limits of IF/IHC
7. Alloantibodies can direct endothelium injury to interact with major
histocompatibility complex (MHC)
8. Increased expression of endothelial transcripts causing endothelium injury.
What is Transplant Glomerulopathy?:
What is c4d negative antibody mediated rejection?
Conclusion,
Thanks Mohamed
SUMMARY OF The Relevance of Complement C4d Staining in Renal Allograft BiopsiesRenal allograft affect by immune response either mediated by macrophages and lymphocytes or by soluble component (antibodies and complement system) which can lead to graft rejection
AMR is a predominant cause of allograft failure
DSA to HLA antigens cause earlier and late graft failure
C4d is afragment of classical complement pathway which is activated by antigen antibody complex
presence of c4d renal biopsy improve diagnosis of AMR
Pathogensis of C4d:
activation of complement of 3 different pathways
complement classical pathway is initiated when c1q with immunoglobulin bind to epitope on the graft endothelium
C1s split c4 into c4a and c4b and then c4b to c4d which is covalent bind to tissue
Clinical of c4d +ve conditions:
1) acute AMR
2) chronic AMR
3) ABO incompatible graft
4) A commendation
C4d -ve status in the following conditions:
Antibody mediated rejection:
Hyper acute AMR:
Chronic AMR:
Mechanism of AMR:
Mixed cellular and humeral rejection:
Transplant glomerulopathy:
Is a chronic form of AMR characterized by
Similarity of morphology of c4d +ve and c4d -ve in form of:
C4d -ve AMR associated with:
accommodation definition:
c4d deposition in PTC in the absence of active rejection with or without DSA ,seen in ABO incompatible graft
Conclusion:
C4d as biomarker has following PROS:
CONS of c4d as biomarker as follows:
Despite all pitfalls c4d is excellent marker for ABMR
Thank You
1.C4d is a foot print of Classical Complement System activation
2. C4d establishes a covalent bond with the tissues and gets deposited for a reasonably long duration in tissues
3. C4d can be the result of activation by Classical pathway, Lectin Pathway and also by C-reactive protein
4. Conditions where c4d can be deposited:
– ABMR – Both Acute & Chronic
– ABO incompatible grafts
– Accommodation
5. Conditions where C4d is Negative:
– TCMR
– False negativity due to technical issues
– NK cell mediated Rejection
– Non Complement Fixing DSA
– Endothelial activation due to Non complement mediated mechanisms
– C4d present but below the threshold of deposition
– Presence of increased expression of Endothelial Transcripts- A marker of poor graft outcome
– Alloantibodies directly interacting with MHC molecules on the endothelium
6. Acute AMR = Exposure of Class 1 & 2Antigens
Chronic AMR = Exposure of Class 2 Antigens
7. C4d Detection in Renal Allograft Biopsies:
– Two Techniques
– IF on Frozen Sections —> Widespread, Strong, Linear Circumferential
– IHC on Paraffin Sections —> Crisp, Linear, Circumferential, Linear with variable intensity
– C4d is significant staining in 1% (Any degree) or more of the PTC for formalin/IHC‐IP, or 10% or more for frozen/frequency IF
8. Prospects of C4d:
– C4d in addition to validated transcripts/classifiers/molecular marker can become potential alternatives or may complement DSA
9. Complement Negative Antibody Mediated Rejection:
– C4d negative AMR was included in 2013 Banff
– How C4d -ve ABMR differs from C4d +ve ABMR: Higher infrarenal endothelial gene expression, Alloantibody expression, Poor Graft Outcomes, Usually after 1 yr post Transplant, Association with Acute on Chronic AMR
10. What is Accommodation:
– C4d deposition in PTCs / absence of Active rejection / DSA +/-
– usually seen in ABOi Tx
11. How much intensity of C4d is Significant ?
– What Banff 2019 States: C4d, linear staining in PTCs or medullary vasa recta by immunofluorescence (IF) on frozen sections of fresh tissue or immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded tissue, scored as 0 (none), 1 (minimal, staining in >0 but <10% of PTCs), 2 (focal, 10%-50% of PTCs), 3 (diffuse, >50% of PTCs). By IF on frozen sections scores ≥2 are considered positive; by IHC on paraffin sections all scores >0 are considered positive
– To simplify, IHC : Any degree of staining ; By IF : > 50 % of staining
– C4d detection by IHC more specific and C4d detection by IF is more sensitive
12. IF Vs IHC:
– IHC: More Specific / Paraffin embedded Tissue / Small Biopsy tissue also Sufficient / Time Consuming / Non specific background Staining
– IF: More sensitive / Frozen sections / Require extra tissue
13. “Pinch of Salt” Facts about C4d: ?? Not as sensitive marker of ABMR as thought initially / Can not be used in ABOi Transplant setting
Thank You
III. The Relevance of Complement C4d Staining in Renal Allograft Biopsies
Please summarise this article
Innate & adaptive immune systems, mediated by cellular (macrophages & lymphocytes) or soluble components (antibodies & complement system), affect graft function & can lead to rejection.
AMR is a predominant cause of allograft failure.
Anti-HLA DSA are significant cause of early & late graft dysfunction & failure.
C4d is a fragment of the classical complement pathway, that is activated by antigen/antibody complexes.
Diagnosis of AMR improves by detection of C4d in renal biopsy.
This review evaluates pathogenesis & current relevance of C4d in AMR.
Pathogenesis of C4d:
Complement is the major constituent of adaptive humoral immunity.
Clinical Relevance of C4d
C4d positivity is seen in:
a. Acute AMR
b. Chronic AMR
c. ABOi grafts
d. Accommodation.
C4d negativity is seen in:
a. TCMR
b. A technical error e.g. type of fixatives, IF versus IHC
c. Antibodies unable to fix the complement
d. Complement independent endothelial activation
e. C4d deposition is very low to be detectable by IF/IHC
f. Alloantibodies to MHC on endothelial cells leading to endothelial injury.
g. Increased expression of endothelial transcripts causing
endothelium injury.
Antibody‑Mediated Rejection
– 30% of all post-transplant rejection events
– 20%–30% graft loss at 1 year if untreated.
– Occur in 3 forms: hyperacute, acute, & chronic
– Presensitization is a primary risk factor for hyperacute & acute rejection.
– Acute AMR associated with HLA Class I & II antigens.
– Chronic AMR is associated with Class II DSA.
– MICA & ABO antigens are other causes of AMR.
Pathology of AMR:
Gross-swollen kidneys(interstitial edema & hemorrhage).
Microscopic – glomerulitis, PTCs, fibrinoid necrosis of arteries, & ATN.
Presence of DSA & C4d deposition PTCs.
Chronic-TG in renal biopsies.
Diagnosis of acute AMR
Based on DSA in plasma, C4d in PTC, clinical & structural features of AMR.
AMR is the chief cause of graft damage specially with positive C4d in PTC combined with high levels of DSA.
Minimum or absent c4d positivity in PTC & absent DSA in plasma make AMR less likely.
Transplant Glomerulopathy:
– A variant of chronic AMR
– Deposition of C4d in a minority of cases.
– Connected to diagnosis of AMR, in the existence of either PTCs or glomerulitis or both as well as with C4d in PTCs.
C4d in allograft Biopsies
C4d score is significant by IF if C4d2 or C4d3 & by IHC C4d >0.
C4d detection by IHC is more specific & C4d detection by IF is more sensitive.
Current Status of C4d
Poor inter-observer reproducibility for C4d IHC
An essential test for AMR(Banff update 2017).
C4d & validated transcripts & molecular marker can be potential alternatives & complements to DSAs in the diagnosis of ABMR.
C4d-negative AMR
Included in Banff 2013.
C4d-positive & C4d-negative AMR have some similarities:
– Varying degrees of glomerulitis
– Peritubular capillaritis
– Frequent TCMR
– Both may occur early or late post-transplantation.
– C4d‑negative AMR have higher intrarenal endothelial gene expression, alloantibodies expression, & poor graft outcomes.
DSA negative AMR:
Characterized by:
– Lack of DSA detection
– Moderate MVI with (g + ptc) scores of ≥2(Banff 2013)
– With or without C4d positivity.
The accommodation:
Characterized by:
– C4d deposition in PTCs
– Absence of active rejection
– With or without DSA positivity
– Seen in ABOi transplantation.
Conclusion
PTC C4d-positive staining in PTC is a diagnostic as
well as a prognostic marker for allograft rejection.
However, from many studies, C4d appears to be a less sensitive marker than initially thought.
Some cons of C4d as a biomarker include:
– Difficulty of interpreting focal staining patterns
– Relatively low sensitivity as a marker for AMR in late renal allograft biopsies
– Its lack of utility as a marker for AMR in ABOi allograft biopsies.
The detection of AMR should best focus on histological features without much reliance on the presence or absence of C4d.
However C4d is still an excellent marker for AMR.
Excellent
Post-transplant graft rejection can happen either due to cellular or antibody-mediated immunological causes. Banff criteria classify rejection into 2 main types, namely antibody mediated rejection (AMR) and cell-mediated rejection (TCMR). AMR caused by donor specific antibodies (DSA) is a predominant cause of graft failure.
Immune complexes activate classical complement pathway, cleaving C4 into C4a and C4b, which in turn bind to hydroxyl or amino group containing molecules via sulfhydryl group giving rise to C4d forming a covalent bond with the tissue. C4 can also be activated through lectin pathway by MBL, H-ficolin and L-ficolin.
C4d positivity is seen in acute AMR, chronic AMR, ABO incompatible transplant and accommodation (C4d deposition in peritubular capillaries without rejection, with or without DSAs).
Rejection with C4d negative stain can be seen with a TCMR, technical error (due to fixatives, IF or IHC), FcRIIA (Fc receptor on NK cell) mediated rejection, non-complement fixing antibodies, very low quantity C4d deposits (below the identification limit of IF/IHC), in conditions with antibodies causing direct endothelial injury (non-HLA antibodies) and with endothelial injury due to increased expression of endothelial transcripts.
AMR (hyperacute, acute or chronic) involves 30% of all rejections and 20-30% of graft loss at 1 year in untreated cases. Past history of sensitization is associated with hyperacute and acute rejection. Acute AMR is associated with both class I and II DSA while chronic AMR is mainly associated with class II DSA. Other antibodies associated with AMR include antibodies against ABO blood group antigens and MICA.
Characteristics of AMR include interstitial edema and hemorrhage causing swollen graft with glomerulits, peritubular capillaritis, fibrinoid necrosis and acute tubular injury, C4d deposition in peritubular capillaries with presence of DSA.
Hyperacute rejection presents within minutes to hours, with a cyanotic and flaccid graft having interstitial edema and widespread cortical necrosis. Acute AMR has C4d in PTC and glomerular capillaries. Chronic AMR shows transplant glomerulopathy (TG).
TG consists of capillary basement membrane splitting or duplication, mesangial expansion of glomeruli, interstitial fibrosis, tubular atrophy and fibrous intimal thickening in arteries, with C4d deposition and without evidence of de novo or recurrent glomerular lesions and TMA. C4d deposition is less in TG.
C4d staining can be done either using immunofluorescence (IF) of frozen sections (significant if ≥10%) or immunohistochemistry (IHC) on formalin preserved paraffin embedded tissue (significant if ≥1%). It has been shown that C4d is not a very sensitive marker for AMR. Microvascular inflammation (MVI) correlated with diffuse C4d positive cases. In Banff 2017 update, for a diagnosis of AMR, in addition to DSA and acute tissue injury, either C4d or transcripts/classifiers/ molecular markers can be used.
Although C4d negative AMR has characteristics similar to C4d positive AMR (frequent TCMR, may occur early or late, varying degree of glomerulitis and peritubular capillaritis), it usually occurs late, has increased alloantibody expression with increased intrarenal endothelial gene expression, is associated with poor graft outcomes due to increased acute on chronic AMR.
C4d is a marker for AMR but has low sensitivity in late graft rejection and is not useful in ABO incompatible transplants. So it is imperative that AMR be reported on the basis of histomorphological features with C4d staining complimenting the biopsy findings.
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The Relevance of Complement C4d Staining in Renal Allograft Biopsies
Introdution
Post kidney transplant, the allograft is affected by many stimulating agents such as antigen‑ antibody‑mediated immunocomplexes and cellular immunity mediated by various cells (macrophages and lymphocytes), which could lead to graft rejection. Based on etiology, the Banff criteria divided the rejection broadly in antibody‑mediated rejection (AMR), and cell‑mediated rejection. AMR is a predominant cause of allograft failure. The clinicians and pathologists now consider antibodies against human leukocyte antigen (HLA) antigens defined as donor‑specific antibodies (DSA) a remarkable cause of early and late graft dysfunction
Complement 4d (C4d) (that is part of component C4) is a fragment of the classical complement pathway, which is activated by immune complexes. The diagnosis of AMR is improved by identification of the complement pieces C4d in renal biopsy, and it has been introduced first under diagnostic criteria of AMR in year 2003.
Pathogenesis of complemente 4d
Complement is an important component of innate and adaptive immunity in our body. Complement is the leading effector constituent of adaptive humoral immunity. There are three different pathways of complement activation, culminating in the deposit of C4d:
– Classical:
– Alternative:
– Lecitin:
Antibody Mediated Rejection
The AMR harms allograft kidney, chiefly in more sensitized patients. AMR accounts for up to 30% of all posttransplant rejection events and 20%–30% graft loss at 1 year in untreated cases. AMR can occur in three forms, namely :
– Hyperacute: The presensitization (blood transfusion, pregnancy, and prior transplant) is a primary risk fator. Occurs within minutes or 24 h after transplantation, mostly due to preexisting DSA present in high titres present in a patient. Grossly, the graft rapidly becomes cyanotic and flaccid. Histomorphology characterized by arteritis, interstitial edema, and extensive cortical necrosis
– Acute: The presensitization (blood transfusion, pregnancy, and prior transplant) is a primary risk factor and acute AMR is most commonly occurs due to expose of MHC Class I and II HLA antigens. C4d is deposited in PTC and glomerular capillaries.
– Chronic rejection: is largely associated with Class II DAS. Histomorphology characterized by transplant glomerulopathy (TG) in renal biopsies. The TG specified as capillary basement membrane duplication or splitting and mesangial expansion of glomeruli, tubular atrophy and interstitial fibrosis, and fibrous intimal thickening in arteries
Diagnosis of acute AMR based on the identification of DSA in plasma, C4 complement component (C4d) in PTC, structural corroboration of AMR, such as kidney injury and graft dysfunction. Mixed cellular and humoral rejection are very hard to examine because it mostly occurs after months and years posttransplantation, and biopsy may show acute and chronic lesion. Here, it is tough to prove that cellular or humoral immune system responsible for the extracellular injury.
Complement 4d Detection in Renal Allograft Biopsies
Two techniques are mostly used for identification of C4d IHC and IF in allograft biopsies of the kidney. IF technique applied to frozen sections whereas IHC technique used to paraffin‑embedded tissue used routinely in various laboratories.
Current Status of Complement 4d
Devadass et al. reported C4d is not very sensitive marker of AMR, as it originally thought. Histologically, moderate to marked microvascular inflammation (MVI) correlated with dispersing C4d positive cases in contrast to focal C4d‑positive cases.
Currently, C4d has become an essential investigation for AMR according to Banff update 2017. In this update, both C4d and validated transcripts/classifiers/molecular marker can serve as potential alternatives and complements to DSAs in the diagnosis of ABMR.
Complement 4d Negative Antibody‑Mediated Rejection
The C4d‑negative AMR is included in Banff 2013 classification. The morphology of C4d‑positive and C4d‑negative AMR having following similar features, however, C4d‑negative AMR morphologically have higher intrarenal endothelial gene expression, alloantibodies expression, poor graft outcomes, usually occur after 1‑year posttransplantation, and associated acute on chronic AMR.
The accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity, mostly seen in ABO‑incompatible graft transplantation.
The TMA (thrombotic microangiopathy) is a complication of AMR in the absence of other causes characterized with fibrin thrombi, fragmented red blood cells, mesangiolysis, and muco‑intimal thickening and injury of small vessels.
Conclusion
The C4d‑positive staining in PTC is a diagnostic marker as well as a prognostic marker for allograft. According to various studies worldwide, C4d appears to be a less sensitive marker than initially thought. The C4d as a biomarker has following PROS :
– (1) provoked an enormous amount of insight in the diagnosis of allograft rejection;
– (2) core diagnostic tool to identify AMR;
– (3) used for vast amount of research into the deposition patterns of C4d in different clinical settings such as pregnancy, thrombotic complications
And has following CONS:
– (1) difficulties of interpreting focal staining patterns;
– (2) relatively low sensitivity of C4d as a marker for AMR in late renal allograft biopsies;
– (3) its lack of utility as a marker for antibody‑mediated injury in biopsies of ABO‑incompatible allografts).
Despite all pitfalls C4d is excellent marker for AMR.
Well done
Antibody mediated rejection occur more in sensitised patients and account 30% of all transplant patients.
History of exposure to pregnancy or transfusion and previous transplant are risk for AMR and usually lead to hyperacute rejection.
Forms of AMR are hyperacute
acute and chronic AMR.
acute AMR due to exposure to MHC class I and class II
chronic AMR associated with exposure to class II DSA.
Pathology of AMR:
grossly swelling of kidney due to interstitial edema and hemorrhage
microscopy finding of AMR shows a. glomerulitis
b. peritubular capillarities
c. fibrinoid necrosis of arteries
d. acute tubular injury
Diagnostic feature of AMR is presence of DSA and C4d deposits in PTC
Causes of C4d positive:
1. acute AMR
2. chronic AMR
3. ABO incompatible
4. Accommodation
causes of C4d negative:
T‐cell‐mediated rejection (TCMR)
b. A technical error of
immunofluorescence (IF) v
or immunohistochemistry
(IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity
g. Alloantibodies can direct endothelium injury to interact
with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing
endothelium injury.
Hyperacute AMR:
Rejection within minutes to 24 hours and occur due to presence of DSA
Grossly graft cyanotic and flaccid
Histomorphological shows arteritis, interstitial edema and extensive cortical necrosis.
Acute AMR:
C4d positive in peritubular capillarities and glomerular capillarities
Chronic AMR:
characters by transplant glomerulopathy manifested by capillary basement membrane duplication and mesangial expansion of glomeruli, tubular atrophy and interstitial fibrosis and fibrous intimal thicking.
Diagnosis of AMR:
1. presence of circulating DSA
2. C4d in peritubular capillary
Mixed cellular and humoral are very hard to examine and occur months to years post transplant in biopsy and may show acute & chronic lesions.
Transplant glomerulopathy:
it’s humoral allograft rejection characteristics by deposition of C4d in peritubular capillarities.
histomorphological manifested by duplication of basement membrane due to accumulation of mesangial cell.
C4d detected by immunoflorecence and immunohistochemistry
by immunoflorescence shows strong linear circumferential peritubular capillary staining in cortex and medulla.
in immunohistochemistry C4d staining linear continuous and diffuse and lying around PTC.
Circulating DSA are associated with C4d positive.
Current status of complement C4d is diagnostic marker for AMR according to banff classification 2017 and associated with moderate to severe micro vascular inflammation.
C4d negative AMR have higher intrarenal endothelial gene expression, alloantibodies and poor graft outcome after one year and associated with acute & chronic AMR.
Accommodation: C4d positive in PTC in absence of acute AMR with or without DSA and seen in patients with ABO incompatible.
Thrombotic microangiopathy is complication of AMR and manifested by fibrin thrombi and fragments RBC and mesangiolysis and muco intimal thickning and vessels injury.
Conclusion:
C4d positive is diagnostic and prognostic marker for AMR but not sensitive.
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The Relevance of Complement C4d Staining in Renal Allograft Biopsies
This review aims to evaluate pathogenesis and current relevance of C4d in AMR
C4d is a degradation product of the classic complement pathway. After an antigen-antibody complex fixes complement, a cascade of events follows with activation of several complement proteins. The complement protein C4 is split into C4a and C4b. C4b is then converted to C4d.
C4d is a complement fragment that is generated through classic pathway activation and is covalently bound to antigen. The results of several studies suggest that peritubular capillary C4d deposition is a marker for acute humoral rejection.
diagnosis of AMR is improved by identification of the complement pieces c4d in renal biopsy, and it has been introduced first under diagnostic criteria of AMR.
Complement 4d is positive in the following conditions.
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation.
Complement 4d is negative in the following situations
a. T‑cell‑mediated rejection (TCMR)
b. A t e c h n ic a l e r r o r l i ke a t y p e of f i x a t ive s ,immunofluorescence (IF) versus immunohistochemistry (IHC)
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC)
h. Increased expression of endothelial transcripts causing endothelium injury
C4d‑negative AMR due to alloantibodies to MHC molecules on endothelial cells, elicit strong responses that include proliferation and activation of intracellular signaling pathways, which leads to endothelium injury.
Also seen in in patients with alloantibody and is an indicator of active antibody‑mediated allograft damage and poor graft outcome.
AMR harms allograft kidney, more sensitized patients. AMR accounts for up to 30% of all posttransplant rejection events and 20%–30% graft loss at 1 year in untreated cases. AMR grades hyperacute, acute, and chronic rejection.
AMR occurs widely in presenstized patient whom previously have blood transfusion, pregnancy ,previous transplantation .
Acute AMR is most commonly occurs due to expose of MHC Class I and II HLA antigens. chronic AMR is largely associated with Class II DSA.
Hyperacute AMR is defined as rejection occurs within minutes or 24 h after transplantation, mostly due to preexisting DSA, Grossly, the graft rapidly becomes cyanotic and flaccid. Histomorphology characterized by arteritis, interstitial edema, and extensive cortical necrosis , In acute AMR, C4d is deposited in PTC
and glomerular capillaries. In chronic AMR, histomorphology characterized by transplant glomerulopathy (TG) in renal biopsies.
Conclusion
C4d‑positive staining in PTC is a diagnostic marker as well as a prognostic marker for allograft.
C4d properties (1) provoked an enormous amount of insight in the diagnosis of allograft rejection, (2) core diagnostic tool to identify AMR, and (3) used for vast amount of research into the deposition patterns of C4d in different clinical settings such as pregnancy, thrombotic complications and CONS as follows: (1) difficulties of interpreting focal staining patterns, (2) relatively low sensitivity of C4d as a marker for AMR in late renal allograft biopsies, and (3) its lack of utility as a marker for antibody‑mediated injury in biopsies of ABO‑incompatible allografts.
Well done
The Relevance of Complement C4d Staining in Renal Allograft Biopsies.
Introduction:
This review aims to evaluate pathogenesis and current relevance of C4d in AMR.
C4d is considered criterion for diagnosis ABMR since 2003,which is degradation of complement factor 4 which is apart from classical complement pathway which activated by immune-complex and stays at the site of complement activation for a longer time, in comparison to other complement pieces.
Pathogenesis of Complement 4d:
When immunoglobulin bounds to epitopes on the graft endothelium , classic complement pathway start to be activated via C1q then C1s splits C4 into C4a and C4b and react with exposing a sulfhydryl group. The ester or amide bond forms with interacting between sulfhydryl group of C4b and nearby molecules containing hydroxyl or amino groups in the tissue.
Cascade become activated with many steps to reach finally to membrane attack complex (membrane‑bound C5b‑9) formed by the addition of C5b component of C5 convertase, that destroys cells.(figure .1)
Clinical Relevance of Complement 4d :
The conditions where complement 4d is positive are:
a. Acute AMR.
b. Chronic antibody‑mediated rejection.
c. A, B, O blood group ABO incompatible grafts.
d. Accommodation.
The state where complement 4d is negative:
a. T‑cell‑mediated rejection (TCMR).
b. A technical error like a type of fixatives,
immunofluorescence (IF) versus immunohistochemistry
(IHC).
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated
rejection.
d. Antibodies unable to fix the complement.
e. Complement independent pathways of endothelial
activation.
f. C4d deposition is a very low in quantity for the
identification limits of IF/IHC.
g. Alloantibodies can direct endothelium injury to interact
with major histocompatibility complex (MHC).
h. Increased expression of endothelial transcripts causing
endothelium injury.
Antibody‑Mediated Rejection:
Acute AMR is most commonly occurs due to expose of MHC Class I and II HLA antigens.
But chronic AMR is largely associated with Class II DSA.
Acute ABMR diagnosed by histological changes(MVI, glomerulitis ,C4d positive and DSA).
Chronic ABMR characterized by chronic histological changes like TG which is duplication of GBM.
Transplant Glomerulopathy:
Histological description which is duplication of the glomerular basement membrane (GBM).
Interstitial fibrosis and tubular atrophy existed along with TG.
Complement 4d Detection in Renal Allograft Biopsies:
Detected by IF or IHC , the first one is more sensitive than IHC and IHC is more specific.
Current Status of Complement 4d:
Currently, C4d has become an essential investigation for AMR according to Banff update 2017.
Complement 4d Negative Antibody‑Mediated Rejection:
The C4d‑negative AMR is included in Banff 2013 classification with same morphological changes of ABMR but no stain for c4d less worse than c4d positive , but also has bad effect on graft survival.
Another form described by BANFF 2013 is DSA negative ABMR with or without c4d.
Conclusion:
C4D is a great marker for ABMR, it has advantages e.g. core diagnostic tool to identify AMR and dis advantages e.g. difficulties of interpreting focal staining patterns and relatively low sensitivity of C4d as a marker for AMR in late renal allograft biopsies , but still is considered diagnostic marker as well as a prognostic marker for allograft.
Well done
The aim of this review study to determine the pathogenesis and the clinical applicability of C4d.
Introduction: Kidney transplantation can be hampered by allograft rejection in particular AMR due to the effect of the antibodies against HLA antigens as well as cellular immune response. C4D is the split product of the classical complement pathway activation by antibodies -antigen immune response during allograft rejection it’s one of the Banff diagnostic criteria of the AMR since 2003
Clinical applicability of C4D
C4D positive
1- Acute and chronic ABMR
2- ABO i Transplantation
3- accommodation of allograft with or without DSA and not associated with allograft injury or rejection
4- Autoimmune diseases like SLE, MGN, MPGN, Ig A nephropathy
C4D negative:
1-Acute cellular rejection (TCMR)
2- technical error by using IF vs IHC staining, IF more sensitive while IHC more specific for C4D staining, also false negative with low level of C4D for detection.
3- presence of non-complement fixing DSA, non-HLA abs
4- complement in dependent pathway of rejection, cytotoxic T cell activation, cellular immune response like in C4D negative ABMR
5- FC NKC receptor induced AR
6-endothilial injury transcription expression which indicate endothelitits in C4 D negative ABMR injury, ENDAT which is carry poor prognosis and lead to progressive graft loss.
Antibody mediated rejection:
The prevalence of AMR up to 30%, and can lead to graft loss in 20-30% after 1year of transplantation, many types of AR including hyper-acute rejection, acute AMR and chronic AMR, acute AMR it due to the performed anti HLA class1 DSA, while chronic AMR is due to class11 anti-HLA DSA.
Histologically AMR characterized by C4D deposition in PTCS, glomerulitis and acute tubular injuries while in Chronic AMR characterized by transplant glomerulopathy (Tg) with the histological features of glomerular capillary basement membrane duplication and splitting, mesangial widening with IFTA, and by EM typical finding of PTC multilayering, chronic intimal arterial thickening and fibrosis
Diagnosis of AMR need the following:
Presence of PTCS, glomerulitis, IHC staining for c4d in addition to the circulatory DSA and clinical evidence of acute graft dysfunction
The effector mechanism of AMR
Direct effect of the DSA on the MHC,
Complement activation (classical complement pathway) with the production of the C4D split product consider the footprint of the antibody’s antigen interaction
Endothelitis with microvascular endothelial injury by cellular FCR NKC.
C4D detection by either using immunohistochemically (IHC) on formalin embedded paraffin sections with positive score >0, and 1% or more positive c4d staining of the PTC the IHC Staining is more specific but less sensitive compared to IF staining while the scoring of C4d is significant if 10% and above by IF on frozen section.
still C4d staining is part of the investigation for AMR
according to Banff update 2017.[44]
but recently with the improvement of the molecular genetic testing both C4d
and validated molecular markers of endothelial transcripts/ molecular marker are essential in the diagnosis of AMR including c4d negative non-complement mediated AMR.
Conclusion:
the C4d‑positive staining in PTC is still an indicative and prognostic marker
for allograft dysfunction and still essential part for the diagnosis of AMR in combination with the typical histological morphology but keep in mind the lower sensitivity of C4D as a marker for AMR in late renal allograft biopsies (non-complement mediated AMR, also in ABO I-transplantation with accommodation and this can be resolved by adding the new molecular genetic biomarkers .
Well done
This article elaborates on different aspects of AMR pathogenesis and relevance of C4d.
AMR is the predominant cause of allograf failure. Antibodies to human leukocyte antigen( HLA)
defined as DSAs are considered a remarkable cause of early and late graft dysfunction.
C4d is part of complement component C4 which is a fragment of classical complement pathway.
Classical complent pathway is activated by immune complexes. C4d make covalent bond with tissue.
It stay at the site of complement activation for a longer time than the other components.
Conditions associated with C4d positivity:
1) Acute AMR
2) Chronic AMR
3) A,B.O incompatible grafts
4) Accommodation.
Negative C4d staining conditions:
1) CMR
2)techn8cal error IF vs IHC.
3) Cytotoxic dependent AMR
4)Complement independent pathway of endothelial activation.
5)Increased expression of endothelial transcripts causing endothelial injury.
AMR:
Account for 30% of all post transplant rejection.
AMR is classified into 3 categories,hyperacute, acute and chronic.
Hyperacute AMR ,
presensitization due to blood transfusion,pregnancy and prior transplantation is an essential risk factor. It occurs within minutes to 24 HOURS, features interstitial edema, artritis and extensive cortical necrosis
Acute AMR :
is due to DSAs against Class I and class II HLA antigens. With activation of complement and deposition of C4d in PTCs and glomerular capillaries.
Chronic AMR :
is due to anti class II DSAs.characterized by TG which features duplication and splitting of GBM,Mesangial expantion IFTA, and fibrous intimal thickening. C4d deposition along GBM presented in minority of cases (6% in one series). It’s associaated with PTC multilayring detected by EM. Glomerulitis and peritubular cap8llaritis are another 2 connection to TG.
Non HLA antigens involved in AMR include
1)MICA(MHC Class I related chain A).
2)ABO blood group antigens.
Effector mechanisms of AMR:
1)Direct effect of DSAs on HLA .
2)Complement fix8ng DSAs.
3)Cellular FCRs.
Diagnostic Criteria of AMR:
1-Microscopic finding of glomerulitis,peritubular capillaritis PTC,fibrinoid necrosis of arteries,and Acute tubular injury.
2-presence of DSAs.
3-C4d deposition in PTCs. Detected by IF or IHC.
Mixed CMR and AMR:
Hard to diagnose, because it occurs months or years post transplantation.
C4d vs MVI:
MVI emerged as another feature reliable to consider in diagnosing AMR.C4d and validated transcript/classifier/molecular marker can serve as potential alternative and complement to DSAs in diagnosing AMR.
C4d negative AMR:
1)Both can occur early or late.
2)vary8ng degree of PTC inflammation,
3)C4d negative AMR have higher intra-renal gene expression,alloantibodies expression,poor graft outcome,and associated with acute on chronic AMR.On the contrary,DSAs negative AMR, ,characterized by absence of DSAs with MVI. And positive or negative C4d.
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Anti HLA DSA are now considered by pathologists and clinicians the culprit of early and late graft injury and failure. C4d is a degradation product of the classical complement pathway, which is activated by antigen antibody complexes.
Complement is an important component of innate and adaptive immunity in our body. Complement system activation is the major contributor of allograft injury in antibody mediated rejection.
The complement system can be activated through three major pathways: classical, lectin, and alternative.
Classical pathway is activated when the C1 complex (C1q, C1r and C1s serine proteasess), binds to the Fc region of complement-fixing antibodies attached to the antigen. Activation of C1r and C1s in turn cleaves C4 and C2 into larger (C4b, C2a) and smaller (C4a, C2b) fragments. This activity underpins the entire complement system terminating in the production of anaphylatoxins and assembly of the MAC and resulting in target cell damage/lysis.
Clinical Relevance of Complement 4d
Following conditions are showing C4d positivity:
a. Acute AMR
b. Chronic antibody mediated rejection
c. A, B, O blood group ABO incompatible grafts
d. Accommodation.
The state where complement 4d is negative
a. T cell mediated rejection
b. A technical error like a type of fixatives, IF versus IHC
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection
d. Antibodies unable to fix the complement
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with MHC
h. Increased expression of endothelial transcripts causing endothelium injury.
AMR can occur in three forms, namely hyperacute, acute, and chronic rejection. Presensitization is a primary risk factor for hyperacute and acute rejection. The diagnosis of AMR requires th
Transplant Glomerulopathy is a morphological change that is mostly seen in humoral rejection and related to immunological etiology. TG is a variant of chronic AMR and characterized by deposition of C4d.
Ultra structure features of TG consist of cytoplasmic vacuolation, broadening of subendothelial space, with the existence of electron dense floccular material, probably due to stiffening of the lamina rara interna. Interstitial fibrosis and tubular atrophy existed along with TG.
Two techniques are mostly used for identification of C4d IHC and IF in allograft biopsies of the kidney. IF technique applied to frozen sections whereas IHC technique used to paraffin embedded tissue.
Currently, C4d has become an essential investigation for AMR according to Banff update 2017.
Excellent
The Relevance of Complement C4d Staining in Renal Allograft Biopsies
C4d is a component of complement factor 4 which is part of the classical pathway.
C4 is cleaved by C1s into C4a and C4 b.
C4d is formed by interaction of sulfhydryl groups of C4b with hydroxyl and amino groups.
C4d makes covalent bonds with tissues and stays longer than other complement factors.
C4d positivity is seen in; Acute AMR, Chronic antibody‑mediated rejection, A, B, O blood group ABO incompatible grafts and Accommodation.
C4d negativity is seen in; TCMR, non- complement fixing antibodies, technical error with fixatives, low level C4d deposition, endothelial injury through non-complement pathways
AMR is a common cause of graft loss.
Pre-sensitization due to previous transplant, pregnancy and blood transfusion.
Acute AMR is due to antibodies against MHC class I and II
Chronic AMR is due to class II DSA
Other non- HLA antigens MICA-A, MICA-B, and ABO antigens.
Pathology of acute AMR; glomerulitis, peritubular capillaritis, fibrinoid necrosis and ATN
In addition to C4d staining in ptc and presence of DSA
Pathology of chronic AMR; Transplant glomerulopathy, peritubular capillary basement membrane multilayering
C4d is detected by either immunofluorescence (IF) on frozen sections or immunohistochemistry (IHC) on paraffin embedded tissue.
C4d is not a very sensitive marker for AMR.
There are also cases of C4d negative AMR.
Well done
C4d staining of the kidney biopsy is important in the diagnosis of renal pathology .
C4d staining can be positive in the following conditions :
In antibody mediated rejection the C4d staining maybe negative due to non-complement binding antibodies ( in which the graft damage is due to antibody mediated cellular cytotoxicity or direct damage to endothelial cells by the anti HLA antibodies ) or due to low quantity of the C4d deposition that not detected by the stain.
C4d staining can be done with either IF or IHC , IF is more sensitive and the results is rapid , whereas IHC is more specific and can be done in cases when no frozen tissue is available but more costly and time consuming
Nice to see your contribution Mujtaba
ABMR : is predominant causes of allograft failure .
DSA a remarkable cause of early and late graft dysfunction .
C4d is fragment of the classical complment pathway , which is activated by Ag _Ab complexes. the dignosis of ABMR by detection of the complement fragment C4d in renal biopsy.
the conditions are showing c4d +ve
acute ABMR
chronic ABMR
A,B, O blood group ABO incompatiable grafts
accommodation
C4d -ve
T -cell mediated rejection
techical error
Fc receptors on NKcell mediated rejection
complement independent pathway of endothial activation
alloantibodies can direct endothilum injury to interact withMHC
increased expression of endothial transcripts causing endothial injury
C4d deposition is avery low in quantity for identification limitsof IF \ IHC
C4d -ve associted with poor graft outcome.
ABMR : occuring in three forms hyperacute , acute and chronic AMR and most commonly occurs due to expose of MHC class 1 and 2
HLA and chronic ccuring with class 2 DSA
pathological finding ABMR kideny become swollen due to interstitial edema and hemmorrage
Microscopic finding of ABMR glomerulitis ,peritubular capillaritis , fibrinoid necrosis of arteries and acute tubular injury.
and present DSA with C4d deposition in peritubular capillaries.Hyperacute AMR occurs within minutes or 24 h after transplantation, caused by preexisting DSA in high titres .Pathological wise characterized by arteritis, interstitial edema, and extensive cortical necrosis.
Acute AMR C4d is deposited in PTC and glomerular capillaries.
Chronic AMR pathologicaly associated with transplant glomerulopathy (TG) specified as capillary basement membrane duplication or splitting and mesangial expansion of glomeruli, tubular atrophy and interstitial fibrosis, and fibrous intimal thickening in arteries.
AMR mechanism :antibodies directed against MHC, Complement fixation, and Cellular FCRs.
mixed cellular and humoral rejection are very hard to examine beacuse it mo0stly occuring after months and years postransplantion
transplant glomerulopathy
Is a morphological change that is mostly seen in humoral rejection and related to immunological etiology and it is a variant of chronic AMR and characterized by deposition of C4d.
TG consists of diffuse multilayering or duplication of the glomerular basement membrane which best seen by jones methenamine silver and periodic acid‑Schiff stain.
Ultra‑structure features of TG consist of cytoplasmic vacuolation, broadening of subendothelial space, with the existence of electron‑dense floccular material.
Interstitial fibrosis and tubular atrophy existed along with TG.
complement c4d detection in renal allograft biopies acute and chronic AMR‑positive C4d stain with IF technique is demonstrated as widespread, strong linear circumferential PTC staining in cortex or medulla, apart from scar or necrotic areas as stated by Banff 2003.
In IHC, C4d staining is crisp, linear, continuous, diffuse, around in the PTC wall, while the strength is slighter , but can have a finely granular pattern in high power.
current status of complement 4d
currently C4d has become an essential investigation for ABMR according to banff update 2017
complement c4d -ve ABMR
c4d -ve ABMR and + ve ABMR having similar features glomerulitis and peritubular capillaritis , frequent TCMR
Both occur early or late posttransplation
note c4d -ve have higher interarenal endothial gene expression with poor graft outcomes.
the accomodation is defined as c4d deposition in PTC in absence of active rejection with or without DSA +ve and mostly seen in ABO incompatible .
TMA is a complication of ABMR in abscence other cause ( fragmented RBC , mesangiolysis and muco intimal thicking and injury of small vessels .
conclusion
C4d staining in PTC is a diagnostic marker .and prognostic marker for marker for allograft.
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Thanks, well done
Complement 4d (C4d) is a fragment of the classical complement pathway, which is activated by immune complexes . C4d makes covalent bond with the tissue. It stays at the site of complement activation for a longer time, in comparison to other complement pieces.
Pathogenesis of complement 4d ;
In the classical pathway of complement kick over of C1 is begun by an interplay of C1q with immunoglobulin bound to epitopes on the graft endothelium. C1s splits C4 into C4a and C4b and react with exposing a sulfhydryl group. C3 convertase (C4bC2a) of classical pathway is formed by combination C4b with enzymatically loose pieces of C2a. C4bC2a (C3 convertase) splits C3 into C3a and C3b. C3b has an active sulfhydryl group which is dative bound and settles in the immediate locality and forms C5 convertase (C4bC2aC3b). C5 convertase split C5 into C5a and C5b. Further, membrane attack complex (membrane‑bound C5b‑9) formed by the addition of C5b component of C5 convertase, that destroys cells.
Following conditions are showing C4d positivity:
a. Acute AMR
b. Chronic AMR
c. ABO incompatible grafts
d. Accommodation.
The following conditions are showing C4d negativity:
a. (TCMR).
b. A technical err or like a type of fixative .
c. Fc receptor (FCR) on NK cells (FcRIIA) mediated rejection.
d. Antibodies unable to fix the complement.
e. Complement independent pathways of endothelial activation
f. C4d deposition is a very low in quantity for the identification limits of IF/IHC
g. Alloantibodies can direct endothelium injury to interact with major. histocompatibility complex (MHC) .
h. Increased expression of endothelial transcripts causing endothelium injury
Anti body mediated rejection ;
The effector mechanisms of AMR;
(1) Direct effects of antibody to MHC .
(2) Complement fixation .
(3) Cellular FCRs.
The DSA produced by plasma cells bind to the endothelium of donor PTC and glomerular capillaries and start the pathological progression of AMR. The C1q is a component of the complement of the classical pathway which ties to the endothelium‑binding DSA, finally leading to graft injury and dysfunction. Diagnosis of acute AMR based on the identification of DSA in plasma, C4 complement component (C4d) in PTC, structural corroboration of AMR, such as kidney injury and graft dysfunction.
Transplant glomerulopathy ;
TG is a variant of chronic AMR and characterized by deposition of C4d. Morphologically, TG consists of diffuse multilayering or duplication of the glomerular basement membrane (GBM).
Complement 4d detection in renal allograft biopsy ;
Two techniques are mostly used for identification of C4d IHC and IF in allograft biopsies of the kidney.
IF technique;
applied to frozen sections . In acute and chronic AMR‑positive C4d stain with IF technique is described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to a consensus at 2003 Banff Conference. C4d detection by IF is more sensitive.
IHC technique;
used to paraffin‑embedded tissue used routinely in various laboratories. In IHC, C4d staining is crisp, linear, continuous, diffuse, and lying around in the PTC wall, while the strength typically is slighter and changeable, but it may have a finely granular pattern in high power. C4d detection by IHC more specific
Complement 4d negative anti body –mediated rejection ;
C4d‑negative AMR morphologically have higher intrarenal endothelial gene
expression, alloantibodies expression, poor graft outcomes, usually occur after 1‑year post transplantation, and associated acute on chronic AMR.
There is another entity of AMR DSA negative AMR, which characterized negative DSA/lack of DSA detection, moderate MVI with (g + ptc) scores of ≥2) as per Banff 2013 with or without C4d positivity.
The accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity, mostly seen in ABO‑ incompatible graft transplantation.
Conclusion ;
The C4d‑positive staining in PTC is a diagnostic marker as well as a prognostic marker for allograft. According to various studies worldwide, C4d appears to be a less sensitive marker than initially thought.
Thanks
Introduction ;
Pathogenesis ;
Clinical relevance of C4d ;
Conditions associated with +ve C4d ; [11,12]
Causes of C4d -ve staining ;[10-14]
AMR ;
TG ;
C4 detection in allograft biopsies ; 2 methods ; linear diffuse deposition[6]
Current status of C4d ;
C4d negative AMR ;
Conclusion ;
Thanks, well done