C4d is a footprint for ABMR. It is found in peritubular capillaries and the vasa recta. It could be detected by either immunofluorescence or immunohistochemistry. Staining could be scored according to staining intensity to
Diffuse (score 3): more than 50% (linear or finely granular)
Focal (score 2): 10-5%
Minimal (score 1): < 10% and more than 0
Negative no staining
Diffuse staining is well correlated with the presence of DSA in the serum and with poorer graft outcomes as it means more complement activation. But in the case of ABOi transplant diffuse staining without evidence of tissue injury could be found with accommodation rather than rejection
-Minimal staining is considered significant by IF.
Wee Leng Gan
2 years ago
No. The risk of AMR in term of pattern of C4d staining is variable. It is common to have histologic evidence of ABMR and a positive DSA but negative C4d staining in the peritubular capillaries. Such condition should treated using the same approach as that for patients with C4d-positive ABMR.
Reference 1)Presentation and Outcomes of C4d-Negative Antibody-Mediated Rejection After Kidney Transplantation.Orandi BJ. Am J Transplant. 2016 Jan;16(1):213-20.
AMAL Anan
2 years ago
C4d stain either minimal , focal , diffuse or negative
Minimal 0-10 % of PTC less significant
Focal 10-50 % OF PTC considered significant
Diffuse > 50 % OF PTC high significant
dina omar
2 years ago
C4d staining is divided into 4 categories:
0: nil
1: minimal : staining >0 , <10% of PTCs indicates : less significant risk of ABMR
2: focal 10%-50% of PTCs indicate : significant risk of ABMR
3: diffuse= >50% of PTCs indicates : Higher significant risk of ABMR
Positive C4d depends on the used method for staining C4d.so:
Positive C4d staining : IF PTCs C4d staining scores equal or > 10%. or ;if in IHC PTCs C4d scores > 0.
Almost 90% of positive C4d are associated with presence of DSA.
C4d +ve but with negative DSA negative rejection can be seen in the following situations: ABO incompatible transplantation, auto-immune disease, and pregnancy.
C4d -ve/ positive DSA can be seen in the following conditions: Technical issues,non-HLA antibodies.
Ahmed Fouad Omar
2 years ago
C4d staining is classified into diffuse, focal, minimal and negative. · Are they all associated with the same risk of AMR? · Substantiate your answer?
o The diagnosis of AMR requires the simultaneous presence of DSA, histo-pathological findings and the linear deposition of C4d in PTC. Additionally, there is a strong relationship between the presence of DSA and C4d staining.
o PTCs linear C4d staining either by immunofluorescence on frozen sections or immunohistochemistry on formalin-fixed, paraffin-embedded tissue can be scored as:
0: No staining
1: Minimal staining which is >0 but <10% of PTCs
2: Focal staining of 10%-50% of PTCs
3: diffuse staining of >50% of PTCs
o Immunofluorescence scores ≥2 are considered positive, whereas; Immunohistochemistry scores >0 are considered positive
o C4d is the immunological foot print for ABMR and complement activation, the greater the complement activation , the higher the risk for acute ABMR.SO,
Diffuse C4d staining is clinically significant and associated with graft loss. It correlates well with the DSA presence.
Focal staining is also considered significant.
Minimal staining is considered significant on IHC but on IF it is not considered significant(IF is very sensitive
o C4d can be negative in presence of DSA, this can occur in:
-Technical issues related to the type of fixative used
-Complement independent AMR
– autoantibodies to AT1R
-FcRIIA (Fc receptor on NK cells)
o Also, We can have C4d positive in the absence of rejection:
– graft accommodation in ABO-incompatible transplantations
– Pregnancy.
– Auto-immune diseases
References
1.Herzenberg A.M. Gill J.S. Djurdjev O. et al.C4d deposition in acute rejection: an independent long-term prognostic factor. J Am Soc Nephrol. 2002; 13: 234-241
2. Batal I, Girnita A, Zeevi A, Saab BA, Stockhausen S, Shapiro R, Basu A, Tan H, Morgan C, Randhawa P. Clinical significance of the distribution of C4d deposits in different anatomic compartments of the allograft kidney. Modern Pathology. 2008 Dec;21(12):1490-8.
Mahmoud Hamada
2 years ago
C4d staing is scored as the following: C4d0 Negative 0% C4d1 Minimal 1<10% C4d2 focal 10-50% C4d3 diffuse > 50% Diffuse PTC C4d staining may occur despite the absence of histologic findings of injury in recipients of ABO-incompatible kidney transplants. on the other hand , Some recipients may have positive DSA and biopsy evidence of ABMR still lack detectable C4d staining.
Hamdy Hegazy
2 years ago
PTCs C4d staining is divided into 4 categories: 0: nil 1: minimal=staining in >0 but <10% of PTCs.–> less significant risk of ABMR 2: focal=10%-50% of PTCs.->significant risk of ABMR 3: diffuse= >50% of PTCs.->Higher significant risk of ABMR Positive C4d depends on the used method for staining C4d. Positive C4d staining is when IF PTCs C4d staining scores equal or above 10%. Positive C4d staining is when IHC PTCs C4d scores more than 0. Almost 90% of positive C4d are associated with presence of DSA.
C4d +ve/ -ve DSA/ negative rejection can be seen in the following situations: ABO incompatible transplantation, auto-immune disease, and pregnancy.
C4d -ve/+ve DSA can be seen in the following conditions: 1-Technical issues, type of staining method. 2-some DSAs have poor complement fixation. 3-non-HLA antibodies like AT1R. 4-Complement independent ABMR.
ahmed saleeh
2 years ago
Linear C4d in PTC either detected by IF (positive if >2) or IHC on frozen paraffin sections (positive if >0).
0: nil
1: (minimal, staining in >0 but 50% of PTCs)
2: (focal, 10%-50% of PTCs)
3: (diffuse, >50% of PTCs)
Diffuse C4d staining is considered significant clinically, and correlates well with DSA presence.
Focal staining is considered significant.
Minimal staining on IHC is considered significant except in IF
C4d negativity in presence of DSA is uncommon, but can be seen due to:
*Technical issues: type of fixative used, different methods of C4d detection.
* Poor complement fixation by some DSAs
* Complement independent AMR
* autoantibodies to AT1R
* FcRIIA (Fc receptor on NK cells)
In a case study: sensitivity of IHC in detecting acute AMR was only 55% and was 23.5% for chronic AMR.
Last edited 2 years ago by ahmed saleeh
MOHAMED Elnafadi
2 years ago
Are they all associated with the same risk of AMR?
NO ACCORDING TO CLASSIFICATION OF C4D STAINING BY Frozen and paraffin section stained by immunoflorescence and immunohistochemistry SO THE GREATER THE PRESCENCE OF AFFECTION THE WORSET THE CONDTION AMR.
0: (none)
1: (minimal, staining in >0 but <10% of PTCs)
2: (focal, 10%-50% of PTCs)
3: (diffuse, >50% of PTCs)
MICHAEL Farag
3 years ago
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 is scored as:
0: (none)
1: (minimal, staining in >0 but <10% of PTCs)
2: (focal, 10%-50% of PTCs)
3: (diffuse, >50% of PTCs)
Immunofluorescence on frozen sections scores ≥2 are considered positive;
Immunohistochemistry on paraffin sections, all scores >0 are considered positive.
There is a strong relationship between the presence of DSA and C4d staining.
Diffuse C4d staining is considered significant clinically and correlates well with DSA presence. Focal staining is also considered significant. Minimal staining on IHC is considered significant, although on IF (as it is very sensitive) it is not considered sensitive.
C4d negativity in presence of DSA is uncommon but can be seen due to:
1) Technical issues: type of fixative used, different methods of C4d detection
2) Poor complement fixation by some DSAs
3) Complement independent AMR
4) AMR due to autoantibodies to AT1R
5) Fc receptor on NK cells
We can have C4d positive WITHOUT rejection in these situations:
ABO-incompatible transplantations and graft accommodation
Systemic Auto-immune disease.
Pregnancy.
Ahmed Omran
3 years ago
C4d linear staining in PTCs or medullary vasa recta by immunofluorescence on frozen sections of fresh tissue or immunohistochemistry on formalin-fixed, paraffin-embedded tissue is 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 immunohistochemistry on paraffin sections all scores >0 are considered positive.
Studies revealed relation between presence of DSA and C4d staining. Sensitivity and specificity of C4d positive staining in a patient with DSA in acute AMR is very high ;more than 90%.
Diffuse C4d staining is clinically significant , and correlates well with DSA . Focal staining is also significant. Also, minimal staining on IHC is considered significant.
C4d negativity in presence of DSA is uncommon, but can be seen in case of :
– Technical issues: type of fixative used, and different methods of C4d detection
– Poor complement fixation by some DSAs
– Complement independent AMR
– AMR due to autoantibodies to AT1R
– Due to Fc RIIA ;Fc receptor on NK cells
Although C4d is a surrogate marker for DSAs, it has low sensitivity in AMR in late graft biopsies ;chronic AMR. It was found that sensitivity of IHC in detecting acute AMR is only 55% and 23.5% for chronic AMR. .
Mohammed Sobair
3 years ago
Are they all associated with the same risk of AMR:
yes.
C4d staining pattern during an early ARE does not predict renal allograft function or
survival over time.
Ref:
Johanna M.Botermans,et al.C4d Staining In Renal Allograft Biopsies with Early Acute Rejection and Subsequent Clinical Outcome.Clin J Am Soc Nephrol. 2011 May; 6(5): 1207–1213.
CARLOS TADEU LEONIDIO
3 years ago
Are they all associated with the same risk of AMR?
No, there´s difference in the risk. The greater the presence of complement, the greater the risk of AMR.
Substantiate your answer?
When assessing the relevance of C4d to allograft failure and accounting for microcirculatory inflamation as a confounder, diffuse C4d was significantly associated with allograft loss, whereas focal C4d was not. Finally, Verghese et al. C4d, did not significantly alter the risk for allograft loss.
REFERENCE:
– A systematic review of the role of C4d in the diagnosis of acute antibody-mediated rejection. Kidney International (2015) 87, 182–194; doi:10.1038/ki.2014.166;
Nasrin Esfandiar
3 years ago
C4d is the immunologic footprint of complement activation and AMR. Staining for C4d is detected by two methods: IHC and IF. If C4d staining in IHC involves more than 10% of PTCs, it is considered positive. But IF is reported positive only if it is diffuse which means involving more than 50% of PTCs. Focal involvement means less than 50% involvement of PTCs by IF C4d staining. C4d is 95% sensitive and 96% specific for DSA. In patients with DSA, positive C4d staining was associated with worse graft function and survival. So, diffuse C4d is a marker for remarkable injury. Some patients have histologic evidence of ABMR and are DSA positive, but are C4d negative in PTCs. In TG only 36% were C4d +, but 73% were DSA positive. Complement independent mechanisms may be involved in these cases. C4d negative ABMR should be treated as ABMR, otherwise they progress to TG and graft loss. But if treated may have better outcome. In addition, ABO incompatible TX, pregnancy and autoimmune diseases will have positive C4d.
amiri elaf
3 years ago
# Peritubular capillary staining was classified as #Diffuse when staining was present in more than half of the microscopic fields examined.
# Staining was referred to as focal when present in 10–50%
# Minimal when 50%, 10–50%, or <10% of the microscopic fields showed the presence of tubular basement membrane C4d staining, respectively. The absence of detectable stain was regarded as negative. To facilitate statistical analysis, C4d staining in the peritubular capillaries and tubular basement membranes was also assigned a numerical score of 0 (negative), 1 (minimal), 2 (focal), or 3 (diffuse). Bowman's capsule staining was qualitatively recorded as present or absent.
Ahuja M, Cohen EP, Dayer AM, et al. Polyoma virus infection after renal transplantation. Use of immunostaining as a guide to diagnosis. Transplantation 2001;71:896–899.
Jamila Elamouri
3 years ago
C4D attaining is classified into diffuse, focal, minimal and negative
The diagnosis of acute AMR requires three components: histologic evidence (glomerulitis, peritubular capillaritis, intimal or transmural/necrotizing arteritis, or acute tubular injury without other apparent cause); evidence of recent antibody interaction with graft endothelium (C4d staining in at least 10% of peritubular capillaries by immunofluorescence on a frozen section or any peritubular capillary C4d staining by immunohistochemistry), and presence of DSA. The C4d score varied from 0 to 3 (negative, minimal, focal and diffuse) depending on the percentage of PTC having a linear staining pattern: no staining = score 0, <10%= score 1 (minimal), 10–50%= score 2 (focal) and >50%= score 3 (diffuse). Microvascular inflammation and injury are not specific to acute AMR. Thus, staining for C4d should be performed on all renal allograft biopsies and routine testing for DSA is also strongly advisable, especially in sensitized patients and whenever AMR is in the differential diagnosis. Although C4d may not be a sufficiently sensitive indicator when compared to microvascular inflammation and DSA class II, that, are more strong indicators of chronic graft outcome.
Negative, minimal and focal/diffuse C4d are associated with simultaneous grades of microcirculation injury and class II DSA. There is notable variation in the C4d status in the first year post-transplant suggesting that ABMR may be a subclinical process, fluctuating in parallel with fluctuations of C4d status.
The correlation between the intensity of fibrosis and deposition of C4d was positive.
There was positivity for IgA, IgM and C3 in groups C4d0, 1, 2 and 3, for IgG and fibrinogen in groups C4d2 and 3, and for C1q in groups C4d1, 2 and 3. C4d3 group showed a higher percentage of fibrosis, with elevated serum creatinine levels, and a positive correlation between fibrosis and intensity of C4d expression.
Linear C4d staining of peritubular capillaries in the absence of evidence of acute tissue injury can be seen in accommodation, which is common in blood group ABO-mismatched transplants.
Granular C4d staining of peritubular capillaries can be seen in lupus nephritis.
(1–4)
1. Collins AB, Schneeberger EE, Pascual MA, Saidman SL, Williams WW, Tolkoff-rubin N, et al. Complement Activation in Acute Humoral Renal Allograft Rejection : Diagnostic Significance of C4d Deposits in Peritubular Capillaries. 1999;(11):2208–14.
2. Seemayer CA, Gaspert A, Nickeleit V, Mihatsch MJ. C4d staining of renal allograft biopsies : a comparative analysis of different staining techniques. 2007;(December 2006):568–76.
3. Corrêa RRM, Machado JR, da Silva MV, Helmo FR, Guimarães CSO, Rocha LP, et al. The importance of C4d in biopsies of kidney transplant recipients. Clin Dev Immunol [Internet]. 2013/07/09. 2013;2013:678180. Available from: https://pubmed.ncbi.nlm.nih.gov/23935649
4. Loupy A, Hill GS, Suberbielle C, Charron D, Anglicheau D, Zuber J, et al. Significance of C4d Banff scores in early protocol biopsies of kidney transplant recipients with preformed donor-specific antibodies (DSA). Am J Transplant Off J Am Soc Transplant Am Soc Transpl Surg. 2011 Jan;11(1):56–65.
manal jamid
3 years ago
– C4d0 No staining of PTCs (0%)
– C4d1 Minimal C4d staining (>0 but<10 % of PTCs) •
– C4d 2 Focal C4d staining (10–50% of PTCs)
– C4d3 Diffuse C4d staining (>50% of PTCs) Diffuse C4d staining in peritubular capillaries (PTCs) during an acute rejection episode (ARE) is the footprint of antibody-mediated rejection. diffuse C4d+ staining during acute rejection is regarded as an inferior prognostic sign. patients with ABMR C4d⁺ had the worst graft survival over 3 years (p = 0.034). Conclusions: This study confirmed that C4d staining in kidney graft biopsies is a clinically useful marker of ABMR, with well-defined clinical and pathological correlations. The impact of C4d deposition in other histologic diagnoses deserves further investigation. Ref Clinical and pathological correlations of C4d immunostaining and its influence on the outcome of kidney transplant recipients. Carpio VN, Rech C, Eickhoff EI, Pegas KL, Edelweiss MI, Gonçalves LF, Manfro RC, Veronese FV.J Bras Nefrol. 2011 Jul-Sep;33(3):329-37. doi: 10.1590/s0101-28002011000300009.PMID: 22042350
nawaf yehia
3 years ago
C4d staining according to the % of PTC involved :
negative
minimal >0 – < 10
focal 10- 50%
diffuse > 50 %
diffuse c4d positivity has the highest correlation with DSA and incidence of ABMR .
C4d +, DSA + , microvascular injury on Bx …. ABMR
C4d – , DSA + , microvascular injury on Bx …. C4d – ve ABMR and this can occur in ( Chronic ABMR with more IFTA,Tg & late graft loss
non complement fixing Ab
aggressive anti rejection Rx before biopsy
technical error)
C4d + , rejection -ve : occur with ABO i transplantation
( here there should be linear ( not circumfencial ) C4d staining , no histological features of A or C ABMR , No molecular evidence ( ENDAT ) of ABMR , NO A or C TCMR changes )
Manal Malik
3 years ago
Are they all associated with the same risk of AMR?
diffuse c4d staining in PTC during acute rejection episode is the footprint of ABMR and also poor prognostic sign,
c4d staining in PTC was evaluated according to the area percentage of positive staining in the renal cortex there are three c4d staining group are made:
1- diffuse positive >50% of PTCs staining.
2-focal 10% to 50% of PTCs
3-negative<10% of PTCs.
staining was performed by both IF and IHC
c4d negative ABMR recently included in banff13 has low incidence,usually present early after transplantation but carries better out come than c4d positive ABMR,,
in diffuse c4d positive is associated higher risk for ABMR than focal or minimal(c4d .
negative), that mean not same risk of ABMR,
although both they have same histologist and serological requirement
support that c4d negative has risk of ABMR is present of chronic ABMR c4d negative histologist finding many studies is more prominent so that mean c4d negative may have same risk of ABMR c4d positive but is over looked.
thus,it appears necessary that diagnostic criteria for ABMR be revised to include c4 d negative lesion
References:
1. Terasaki PI, Cai J. Human leukocyte antigen antibodies and chronic rejection: from association to causation. Transplantation 2008; 86:377– 383.
2. Colvin RB. Antibody-mediated renal allograft rejection: diagnosis and pathogenesis. J Am Soc Nephrol 2007; 18:1046–1056.
Mohamed Ghanem
3 years ago
Staining of C4d either by IF of frozen sections or IHC of paraffin-embedded sections classified into : all sections. C4d staining in PTCs was evaluated according to the area percentage of positive staining in the renal cortex as described in the Banff criteria. Three C4d staining groups were made: Negative no staining minimal (<10% of PTCs) focal (10% to 50% of PTCs), and diffuse positive (>50% of PTCs) staining Negative C4d : C4d negative ABMR better outcome but need aggressive treatment like positive c4d ABMR
The state where complement 4d is negative
The following conditions are showing C4d negativity
a. T‑cell‑mediated rejection (TCMR)
b. technical errors of fixation
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 Focal staining : it may represent an evolving AMR. However, a focal staining pattern is frequently observed during the resolution of AMR episode
diffuse positive (>50% of PTCs) staining : Indicate the presence of Alloantibodies directed against HLA antigen with complement-dependent pathway With more prevalent in : a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO-incompatible grafts
d. Accommodation.
However Botermans et al retrospective study showed that C4d staining is not related to clinical outcomes in this large cohort of first histologically proven early (<6 months),
Reference : The Relevance of Complement C4d Staining in Renal Allograft Biopsies Anju Khairwa*
C4d is a degradation product of the classic complement pathway. IHC is less sensitive than immunofluorescent detection methodsC4d scoring
0: (none)
1: (minimal, staining in >0 but <10% of PTCs)
2: (focal, 10%-50% of PTCs)
3: (diffuse, >50% of PTCs)
Presence of positive C4d associated with presence of DSA and most probably AMR . We can have +ve C4d in autoimmune disease ,pegnancy and ABO incompatible transplantation which consider as false positive
Negative C4d cannot rule out AMR it can be du to :
a- antibodies didn’t fix the complement
b- Fc -portion in NK cell
c- AMR du to auto-antibodies
d- Technical error desensitized patients with AMR have similar outcomes regardless of C4d staining .
Balaji Kirushnan
3 years ago
C4d is complement degradation product of C4b which is formed in the classical pathway. It has sulfahydryl groups which have strong covalent binding into the endothelium of the vasa recta and peri tubular capillaries. C4d is considered the foot print of ABMR representing deposition after HLA antigen and anti HLA antibody.
C4d staining can be done in renal allograft biopsies by IF (immunofluorescence) by using either mono clonal or polyclonal antibodies or by IHC (immunohistochemistry) from formalin embedded tissues. Banff Classification recognized C4d staining as a diagnostic marker of ABMR in 2003. In the 2007 Banff classification C4 d staining was scored and listed as follows
0 – C4d negative in PTC and medullary vasa recta
1 – C4d Positive >0 – <10% in PTC and medullary vasa recta
2 – C4d Positive – Focal – >10% in PTC and medullary vasa recta
3 – C4d Positive – Diffuse >50% in PTC and medullary vasa recta
2013 Banff conference has recommended that atleast 10 percent of C4d staining is recommended to define ABMR.
Diffuse C4d positivity is associated with higher levels of DSA and more severe ABMR than focal
C4d negative ABMR is associated with low levels of DSA which are usually class II DSA and are associated with late onset ABMR
C4d negative ABMR is also seen with non HLA mediated ABMR like anti endothelin or ATR1 receptor antibody mediated ABMR, FC receptor NK cell mediated ABMR, non complement binding DSA, low level of complement binding DSA, technique failure (improper methods)
C4d has been associated with glomerular deposits in diseases like Lupus nephritis, membranous nephropathy, minimal change disease. They signify classical pathway activation of complement
fakhriya Alalawi
3 years ago
C4d, a complement split product, is regarded as a footprint of AMR. Peritubular capillary C4d staining was classified as diffuse when staining was present in more than half of the microscopic fields examined. Staining was referred to as focal or minimal when present in 10–50% or <10% of the microscopic fields, respectively. The absence of detectable staining was regarded as negative.
Botermans JM et al, found Sensitivity (95%) and specificity (96%) of diffuse C4d staining in PTCs for the presence of DSAs is high. Similarly, other studies have found a strong relation between diffuse C4d staining of PTCs and the presence of DSAs. However, he found no difference in the occurrence of steroid resistance or graft survival for patients who in retrospect showed a C4d+ or a C4d− graft rejections and he concluded that the C4d staining pattern during an early graft rejections does not predict renal allograft function or survival over time.
On the other hand, Batal I et al, found that diffuse C4d was associated with an inferior outcome even in the absence of demonstrable donor-specific antibodies. However, Rosales IA et al, in a study describing serial allograft biopsies of pre-sensitized patients, found that the C4d was initially negative and later was noted to be positive. Similarly, others found that Antibody levels and C4d deposition can fluctuate with time and may be a less active process at the time of biopsy.
References:
1. Botermans JM, de Kort H, Eikmans M, Koop K, Baelde HJ, Mallat MJ, Zuidwijk K, van Kooten C, de Heer E, Goemaere NN, Claas FH. C4d staining in renal allograft biopsies with early acute rejection and subsequent clinical outcome. Clinical Journal of the American Society of Nephrology. 2011 May 1;6(5):1207-13.
2. Rosales IA, Colvin RB. Acute Antibody-Mediated Rejection. InKidney Transplantation, Bioengineering and Regeneration 2017 Jan 1 (pp. 475-487). Academic Press.
3. Colvin RB, Chang A. Diagnostic pathology: kidney diseases. Farris AB, editor. Amsterdam: Elsevier; 2016.
4. Batal I, Girnita A, Zeevi A, Saab BA, Stockhausen S, Shapiro R, Basu A, Tan H, Morgan C, Randhawa P. Clinical significance of the distribution of C4d deposits in different anatomic compartments of the allograft kidney. Modern Pathology. 2008 Dec;21(12):1490-8.
Dalia Ali
3 years ago
Diffuse C4d staining in peritubular capillaries (PTCs) during an acute rejection episode (ARE) is the footprint of antibody-mediated rejection. In current clinical practice, diffuse C4d+ staining during acute rejection is regarded as an inferior prognostic sign.
C4D is specific but not sensitive marker of ABMR
C4D negative is less common than C4D + cases
C4d+ve AMR: more often seen in patients with DSA against HLA class I, or both class I and II.
C4D positive ABMR occurs earlier and more severe than C4D -ve
Banff recommendations.
C4d
diffusely positive if ≥50% of PTCs
focal positive if <50%
negative if very little (<10%) or no staining was seen in accordance
C4d-negative antibody-mediated rejection in renal allografts is Less severe than C4d-positive AMR (potential for causing transplant glomerulopathy, IF/TA, and graft loss)
But If not treated, it will result in all of these. So , Aggressive treatment of c4d negative ABMR is recommended and anti Complement treatment is not effective
1-Johanna M Botermans, Hanneke de Kort, […], and Marian C. van Groningen. C4d Staining In Renal Allograft Biopsies with Early Acute Rejection and Subsequent Clinical Outcome. American Society of Nephrology.
2-Renal Allograft Rejection PART -III Tarek Medhat Abbas, MD Prof of Nephrology Mansoura University
Zahid Nabi
3 years ago
In normal kidneys, C4d is detectable in the glomerular mesangium and at the vascular pole. Deposition of C4d in the PTCs has only been described in renal allografts and represents complement activity directed against donor antigen
Criteria for C4d staining is linear & circumferential deposition in at least 25% of cortex & medulla excluding necrotic tissues.
0- absent
1- minimal (50%)
C4d staining in PTC is highly specific for presence of DSA, so when deposition is diffuse mean there is strong correlation with anti-HLA Abs with poor graft outcome however C4d staining in ABO-i indicates ‘graft accommodation’ rather than AMR.
Ahmed Abd El Razek
3 years ago
Introduction:
detection of C4d, a single complement degradation product that can independently predict unfavorable graft outcome. C4d participates in the classic pathway of complement activation. Activated C4 is cleaved to C4a and C4b. C4b binds to an amino or hydroxyl group and is then converted to C4d, which can covalently bind with peritubular capillary basement membrane.
Risk of AMR is :
Linear or finely granular peritubular capillary staining was classified as diffuse when it was seen in more than 50% of microscopic fields available for evaluation, focal when present in 10–50% of biopsied tissue, and negative if the staining did not even reach
that threshold.
diffuse pattern is highly correlated with circulating DSA and highest incidence of AMR followed by focal pattern.
In terms of extent of staining, with IF, Banff Lesion Score C4d ≥ 2 is considered positive and a criterion for antibody interaction with tissue and as equivalent to DSA.
C4d0—No staining of PTC and medullary vasa recta (0%).
C4d1—Minimal C4d staining (>0 but <10% of PTC and
medullary vasa recta).
C4d2—Focal C4d staining (10-50% of PTC and medullary
vasa recta).
C4d3—Diffuse C4d staining (>50% of PTC and medullary
vasa recta).
C4D negative samples could be false negative ( eg technical errors and may need another sample, or could be T cell mediated with scarce antibody level to be detected)
Reference Guide to the Banff Classification of Renal Allograft Pathology, Transplantation: November 2018 – Volume 102 – Issue 11 – p 1795-1814
mai shawky
3 years ago
C4d staining in PTCs either identified by IF on frozen sections of fresh tissue or IHC on, paraffin-section and is scored as:
0: (none)
1: (minimal, staining in >0 but <10% of PTCs)
2: (focal, 10%-50% of PTCs)
3: (diffuse, >50% of PTCs)
Immunofluorescence on frozen sections scores ≥2 are considered positive;
Immunohistochemistry on paraffin sections, all scores >0 are considered positive.
Diffuse C4d staining is considered significant clinically and correlates well with DSA presence. Focal staining is also considered significant. Minimal staining on IHC is considered significant, although on IF (as it is very sensitive) it is not considered sensitive. There is a strong relationship between the presence of DSA and C4d staining.However, some exceptions exist:
· C4d negative AMR (+ve DSA) may be due to :
1. Technical errors
2. Different method of c4d detection (IF vs IHC).
3. Very low amount of c4d (difficult detection).
4. Poor complement fixation by DSA
5. Complement independent pathway AMR or antibody dependent cell mediated cytotoxicity.
6. Antibodies against Angiotensin II type 1 receptors
7. fc receptors of Nk may have a role c4d negative AMR.
8. Increased expression of endothelial transcripts causing endothelium injury.
9. Sure pure TCMR will have -ve c4d.however, mixed rejection may be either c4d +ve or -ve.
· C4d +ve staining without AMR (-ve DSA):
o Not every c4d +ve means AMR: (sometimes accommodation to antibodies occurs so no immunologically mediated damage occur).
o ABO incompatible transplantation will have +ve c4d.
o Immune mediated kidney disease as lupus nephritis or anti phospholipid syndrome (either recurrent in the graft or infrequently present in donor and not detected). However, mostly here the c4d deposits will be mesangial or glomerular (not in peritubular capillaries).
Last edited 3 years ago by mai shawky
Theepa Mariamutu
3 years ago
C4d staining is classified into diffuse, focal, minimal and negative
Are they all associated with the same risk of AMR?
Substantiate your answer?
C4d linear staining in pertitubular capillary vasa recta by IF on Frozen sections of fresh tissues or IHC on formalin fixed, paraffin -embedded tissues is scored according to:
1- minimal staining >0 but 10% but 50% staining
IF C4d2 and more considered positive but IHC more than 0 is considered positive
Some conditions associated with diffuse C4d positivity without ABMR:
1- ABOi- due to graft accommodation
2- Pregnancy
3- Autoimmune diseases such as SLE
ABMR is diagnosed by presence of DSA, histological features that suggestive and C4d in PTC
Diffuse C4d is strongly associated with capillarities and TG which are the features of ABMR
C4d positivity in lupus nephritis is a bit tricky, it can be ABMR which need to be considered
C4d negative
C4d negative could be due to complement independent form of ABMR. Study by loupy showed that C4d or capillaritis in protocol biopsy determines the progression to TG and capilaritis without C4d also had higher rate of TG progression.
C4d negative ABMR usually overlooked and never been treated and study by Haas and Mirocha showed that C4d negative ABMR had higher rate of progression compared to treated C4d positive ABMR
References
1.Herzenberg A.M. Gill J.S. Djurdjev O. et al.C4d deposition in acute rejection: an independent long-term prognostic factor.J Am Soc Nephrol. 2002; 13: 234-241
2.Loupy A. Hill G.S. Suberbielle C. et al.Significance of C4d Banff scores in early protocol biopsies of kidney transplant recipients with preformed donor-specific antibodies (DSA). Am J Transplant. 2011; 11: 56-65
3.Haas M. Mirocha J. Early ultrastructural changes in renal allografts: correlation with antibody-mediated rejection and transplant glomerulopathy Am J Transplant. 2011
Last edited 3 years ago by Theepa Mariamutu
Sahar elkharraz
3 years ago
C4d staining is Frozen and paraffin section stained by immunoflorescence and immunohistochemistry.
C4d staining is not diagnostic for AMR without histological feature of acute or chronic AMR.
There’s some conditions associated with C4d positive without histological feature like
– [ ] ABO incompatible
– [ ] Pregnancy
– [ ] Autoimmune disease
C4d staining is classified to
Diffuse > 50% peritubular capillarities and it’s associated with high level of DSA and it’s gold stander diagnosis of AMR and it’s footprints AMR.
Focal C4d staining is 10 to 50% peritubular capillarities
Minimal C4d staining less than 10% peritubular capillarities.
Negative C4d staining means no peritubular capillarities.
References:
Merk Hass, The significance of C4d staining with minimal histologic abnormalities; natural library of medicine curr open organ transplant: 2010 Feb.
Johanna M Botermans, et al; C4d Staining In Renal Allograft Biopsies with Early Acute Rejection and Subsequent Clinical Outcome: Clin J Am Soc Nephrol. 2011 May; 6(5): 1207–1213.
Nazik Mahmoud
3 years ago
C4d scoring
Scoring of C4d staining is based on the percentage of
stained tissue on IF/IHC that has a linear, circumferential staining pattern in PTC : C4d0—No staining of PTC (0%).
C4d1—Minimal C4d staining0-10%
C4d2—Focal C4d staining 10-50%
C4d3—Diffuse C4d staining >50%
Presence of positive C4d associated with presence of DSA and most probably AMR except in autoimmune disease and transplant a cross ABO incompatible which consider here as false positive,on the other hand negative C4d cannot rule out AMR because there’s many antibodies didn’t fix the complement
Mahmud Islam
3 years ago
As Cohen, Danielle et al. described in “Pros and cons for C4d as a biomarker.”
C4d is scored semiquantitatively in four categories:
No C4d staining (0% of (peritubular) capillaries)
Minimal C4d staining (0–10% of (peritubular) capillaries)
Focal C4d staining (10–50% of (peritubular) capillaries)
Diffuse C4d staining (>50% of (peritubular) capillaries)
Immunofluorescence is a more sensitive d than immunohistochemistry but we need a fresh frozen section. In IHC the positivity carries more importance when detected.
Reference: Kidney international vol. 81,7 (2012): 628-39. doi:10.1038/ki.2011.497
There is debate about the importance of cd negative AMR but we know that may be due to early stage. so In case of cd4 negative AMR as AMR may be present without effect on eGFR we need to correlate histologic findings with serial monitoring of GFR and DSA level
Mohamed Mohamed
3 years ago
4. C4d staining is classified into diffuse, focal, minimal and negative. Are they all associated with the same risk of AMR? Substantiate your answer?
The diagnosis of AMR requires the simultaneous presence of DSA, characteristic histopathological findings & linear deposition of C4d in PTC. Most centers include routine C4d staining in diagnostic pathology evaluation of all renal allograft biopsies.
More than 70–80% of ABO-i grafts showed diffuse C4d positivity.
In contrast to conventional transplants where a diffuse C4d stain is strongly associated with histological abnormalities such as capillaritis & TG, the ABO-I kidneys as a rule show diffuse C4d positivity without histological tissue injury. C4d staining in ABO-i indicates ‘graft accommodation’ rather than AMR.
PTC C4d staining was tested in many forms of GN, where PTC C4d staining was virtually never observed.
The only exception was lupus nephritis, in which granularPTC staining has been rarely described, which should be kept in mind when a diagnosis of AMR in a transplanted SLE patient is considered.
C4d-negative AMR:
Molecular studies showed a complement-independent form of AMR or C4d-negative AMR, in which C4d is obviously not helpful as a diagnostic tool. Loupy et al. showed that C4d or capillaritis in 3-month protocol biopsies were risk factors for later TG, & capillaritis was predictive even in the absence of C4d. Haas & Mirocha investigated patients with DSAs who had a biopsy during the first 3 months posttransplant. Patients with a C4d-negative biopsy who were not treated for AMR had a higher rate of progression to TG than those who were treated for AMR post-biopsy.
References 1.Herzenberg A.M. Gill J.S. Djurdjev O. et al.C4d deposition in acute rejection: an independent long-term prognostic factor.J Am Soc Nephrol. 2002; 13: 234-241 2.Loupy A. Hill G.S. Suberbielle C. et al.Significance of C4d Banff scores in early protocol biopsies of kidney transplant recipients with preformed donor-specific antibodies (DSA). Am J Transplant. 2011; 11: 56-65 3.Haas M. Mirocha J. Early ultrastructural changes in renal allografts: correlation with antibody-mediated rejection and transplant glomerulopathy Am J Transplant. 2011
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 is scored as: 0: (none) 1: (minimal, staining in >0 but <10% of PTCs) 2: (focal, 10%-50% of PTCs) 3: (diffuse, >50% of PTCs) Immunofluorescence on frozen sections scores ≥2 are considered positive; Immunohistochemistry on paraffin sections, all scores >0 are considered positive. There is a strong relationship between the presence of DSA and C4d staining. Diffuse C4d staining is considered significant clinically and correlates well with DSA presence. Focal staining is also considered significant. Minimal staining on IHC is considered significant, although on IF (as it is very sensitive) it is not considered sensitive. C4d negativity in presence of DSA is uncommon but can be seen due to: 1) Technical issues: type of fixative used, different methods of C4d detection 2) Poor complement fixation by some DSAs 3) Complement independent AMR 4) AMR due to autoantibodies to AT1R 5) Fc receptor on NK cells
We can have C4d positive WITHOUT rejection in these situations:
ABO-incompatible transplantations and graft accommodation
Diffuse C4d staining in peritubular capillaries (PTCs) is considered as footprint of antibody-mediated rejection ,but is regarded as an inferior prognostic sign as C4d staining is not related to clinical outcome in the histologically proven early Acute rejection.(1)
Focal PTC C4d+ staining detected via IP was shown to be associated with circulating DSA and AMR . Combined focal and diffuse PTC C4d+ staining by IP has a similar sensitivity when compared with the IF method . Isolated glomerular C4d+ staining was detected in lupus nephritis and other glomerular diseases of native kidney biopsies and in transplant glomerulopathy of allograft biopsies , but its association with DSA and AMR in kidney transplant recipients is unclear.
Although diffuse C4d+ staining of PTC in ABO-compatible kidney allografts is strongly associated with AMR, C4d+ staining of PTC is common in ABO-incompatible kidney transplant recipients and may be seen in up to 80% of protocol biopsies without any acute rejection findings , suggesting that the presence of C4d on endothelial cells might play a role in accommodation of the graft in ABO-incompatible kidney transplantation and is not associated with AMR. It was reported that biopsies with minimal PTC C4d staining demonstrate gene expression profiles and Banff injury scores similar to focal/diffuse PTC C4d+ biopsies, are associated with circulating DSAs, acute and chronic AMR , therefore are a valid marker for AMR; however, isolated glomerular C4d+ biopsies do not have the gene expression profiles or association with circulating DSAs as seen with all PTC C4d+ groups and are not a marker for AMR.(2)
Graft dysfunction with diffuse positivity for C4d staining require specific anti-humoral therapy. Cases presenting with minimal and focal positivity for C4d showed improved graft function with pulse dose steroids and change in immunosuppression. C4d detection is a useful adjunct in the diagnosis of humoral responses in renal transplant patients presenting with graft dysfunction and helps in guiding clinical decisions for achieving long-term graft survival.(3)
C4d-negative AMR described in a chronic, but not in acute settings; led to using endothelial transcripts combined with DSA as a marker for AMR. Biopsies with high expression of these endothelial transcripts in combination with circulating DSA showing concurrent histopathological lesions of AMR had poor outcomes. Patients with a C4d-negative biopsy who were not treated for AMR had a higher rate of progression to transplant glomerulopathy than those who were treated for AMR post-biopsy.(4)
Reference
1-Botermans J M et al.C4d Staining In Renal Allograft Biopsies with Early Acute Rejection and Subsequent Clinical Outcome.Clin J Am Soc Nephrol.2011 May; 6(5): 1207–1213.
2-Hayde, Nicole ; Bao, Yi; Pullman, James; Ye, Bin ‘; Calder, Brent R.; Chung, Monica; Schwartz, Daniel; Alansari, Ahmed; de Boccardo, Graciela; Ling, Min; Akalin, Enver . The Clinical and Molecular Significance of C4d Staining Patterns in Renal Allografts, Transplantation Journal: February 27, 2013 – Volume 95 – Issue 4 – p 580-588
3-Guduru SL, Kuruvilla S, Abraham G, Mathew M, Saravanan S. Diagnostic efficacy of C4d immunostaining in the detection of the humoral component of renal allograft rejection and therapeutic implications. Indian J Pathol Microbiol 2009;52:345-8.
4-Cohen D etal . Pros and cons for C4d as a biomarker. Kidney Int. 2012 April ; 81(7): 628–639.
Scoring of the C4d expression in renal allograft biopsies
Biopsies were scored into four categories for the circumferential expression of C4d along PTC in the unscarred renal cortex:
1.Diffuse expression: >50% of PTC positive.
2. Focal expression: staining of at least 10 PTC and <50%.
3. Focal minimal expression: staining of at least 3 but less than 10 PTC.
4. Negative: staining of less than three PTC or completely negative.
-c4d positivity is correlated with ABMR, and even a focal c4d staining in the specimen is well-associated with active humoral rejection.
-Diffuse peritubular capillary C4d deposition in renal allograft biopsies is associated with donor-specific antibodies (DSA),ABMR and graft failure.
-focal C4d had similar impact on graft survival as diffuse pattern.
-focal minimal has minimal risk of ABMR.
-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 References:
1.Christian A. Seemayer, Ariana Gaspert et al. C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques. Nephrology Dialysis Transplantation, Volume 22, Issue 2, February 2007, Pages 568–576.
2-Diana Taheri, Ardeshir Talebi, Maryam Taghaodi et al. Pathological diagnosis of antibody-mediated rejection in renal allograft without c4d staining, how much reliable? ,Adv Biomed Res. 2012; 1: 40.
can you think of any variation in interpretation for ABOi transplants!
Abdul Rahim Khan
3 years ago
C4d staining in renal allograft biopsies can be –
1-Diffuse- >50 %of PTC are positive
2-Focal- 10-50% PTC are positive.
3- Focal minimal- Staining of at least 3 but <10 PTC
4-Negative- Completely negative or <3PTC
Significance of C4d staining
Focal staining can be associated with DSA and ABMR while diffuse staining carries high risk of ABMR due to tissue injury and microvascular inflammation. Focal minimal signifies chronic disease. In C4d negative cases the mechanism of injury is through NK cells and neutrophils and DSA are positive. Negative C4d staining if associated with High expression of ENDATs, DSA and microvascular inflammation are enough for AMR diagnosis.
Reference:
Johanna M Botermans et al. C4d Staining In Renal Allograft Biopsies with Early Acute Rejection and Subsequent Clinical Outcome. Clin J Am Soc Nephrol. 2011 May; 6(5): 1207–1213.
Decent arguments and as you implied C4d shows that the complement system is involved. A step further medicine is advancing, we know that complement targeted therapies shall be a primal future treatment option and a marker such as C4d will identify patients susceptible to those treatments
kumar avijeet
3 years ago
C4d staining read as-
1.No c4d staining-0% of ptc
2.minimal c4d-1-10% of ptc
3.focal c4d-10-50% of ptc
4.diffuse c4d->50% of ptc
These all grading are not associated with same risk of AMR.
C4d staining done by two methods-
A.IF study is more sensitive
B.IHC study is more specific
So,when IF study used focal and diffuse c4d staining marked as one of the character for ABMR, but when IHC study is used minimal, focal,diffuse c4d staining used as criteria in presence of histological injury and DSA.So the method of c4d staining defines the risk, but obviously in presence of other criteria like histological injury +presence of DSA.
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 is scored as: (1)
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.
Various studies have shown that there is a relation between the presence of DSA and C4d staining. The sensitivity and specificity of C4d positive staining in a patient with DSA (in acute AMR) is very high (more than 90%). (2-6)
Diffuse C4d staining is considered significant clinically, and correlates well with DSA presence. Focal staining is also considered significant. Minimal staining on IHC is considered significant, although on IF (as it is very sensitive) it is not considered sensitive. C4d negativity in presence of DSA is uncommon, but can be seen due to: (7) 1) Technical issues: type of fixative used, different methods of C4d detection 2) Poor complement fixation by some DSAs 3) Complement independent AMR 4) AMR due to autoantibodies to AT1R 5) Due to FcRIIA (Fc receptor on NK cells)
Although C4d is a surrogate marker for DSAs, it has low sensitivity in AMR in late graft biopsies (chronic AMR). (8) In fact, one study found that the sensitivity of IHC in detecting acute AMR was only 55% and was 23.5% for chronic AMR. (9)
Reference:
1) Loupy A, Haas M, Roufosse C, Naesens M, Adam B, Afrouzian M, Akalin E, Alachkar N, Bagnasco S, Becker JU, Cornell LD, Clahsen-van Groningen MC, Demetris AJ, Dragun D, Duong van Huyen JP, Farris AB, Fogo AB, Gibson IW, Glotz D, Gueguen J, Kikic Z, Kozakowski N, Kraus E, Lefaucheur C, Liapis H, Mannon RB, Montgomery RA, Nankivell BJ, Nickeleit V, Nickerson P, Rabant M, Racusen L, Randhawa P, Robin B, Rosales IA, Sapir-Pichhadze R, Schinstock CA, Seron D, Singh HK, Smith RN, Stegall MD, Zeevi A, Solez K, Colvin RB, Mengel M. The Banff 2019 Kidney Meeting Report (I): Updates on and clarification of criteria for T cell- and antibody-mediated rejection. Am J Transplant. 2020 Sep;20(9):2318-2331. doi: 10.1111/ajt.15898. Epub 2020 May 28. PMID: 32463180; PMCID: PMC7496245. 2) Troxell ML, Weintraub LA, Higgins JP, Kambham N. Comparison of C4d immunostaining methods in renal allograft biopsies. Clin J Am Soc Nephrol. 2006 May;1(3):583-91. doi: 10.2215/CJN.00900805. Epub 2006 Mar 29. PMID: 17699262. 3) Böhmig GA, Exner M, Habicht A, Schillinger M, Lang U, Kletzmayr J, Säemann MD, Hörl WH, Watschinger B, Regele H. Capillary C4d deposition in kidney allografts: a specific marker of alloantibody-dependent graft injury. J Am Soc Nephrol. 2002 Apr;13(4):1091-1099. doi: 10.1681/ASN.V1341091. PMID: 11912271. 4) Mauiyyedi S, Crespo M, Collins AB, Schneeberger EE, Pascual MA, Saidman SL, Tolkoff-Rubin NE, Williams WW, Delmonico FL, Cosimi AB, Colvin RB. Acute humoral rejection in kidney transplantation: II. Morphology, immunopathology, and pathologic classification. J Am Soc Nephrol. 2002 Mar;13(3):779-787. doi: 10.1681/ASN.V133779. PMID: 11856785. 5) Collins AB, Schneeberger EE, Pascual MA, Saidman SL, Williams WW, Tolkoff-Rubin N, Cosimi AB, Colvin RB. Complement activation in acute humoral renal allograft rejection: diagnostic significance of C4d deposits in peritubular capillaries. J Am Soc Nephrol. 1999 Oct;10(10):2208-14. doi: 10.1681/ASN.V10102208. PMID: 10505698. 6) Sapir-Pichhadze R, Curran SP, John R, Tricco AC, Uleryk E, Laupacis A, Tinckam K, Sis B, Beyene J, Logan AG, Kim SJ. A systematic review of the role of C4d in the diagnosis of acute antibody-mediated rejection. Kidney Int. 2015 Jan;87(1):182-94. doi: 10.1038/ki.2014.166. Epub 2014 May 14. PMID: 24827778. 7) Nigam LK, Vanikar AV, Kanodia KV, Patel RD, Suthar KS, Patel HV. C4d-negative antibody-mediated rejection: A pathologist’s perspective and clinical outcome. Saudi J Kidney Dis Transpl. 2018 Jan-Feb;29(1):39-49. doi: 10.4103/1319-2442.225206. PMID: 29456206. 8) Etta PK. C4d staining and antibody-mediated rejection in renal transplantation: Current status. Indian J Transplant 2020;14:197-201
9) Devadass CW, Vanikar AV, Nigam LK, Kanodia KV, Patel RD, Vinay KS, Patel HV. Evaluation of Renal Allograft Biopsies for Graft Dysfunction and Relevance of C4d Staining in Antibody Mediated Rejection. J Clin Diagn Res. 2016 Mar;10(3):EC11-5. doi: 10.7860/JCDR/2016/16339.7433. Epub 2016 Mar 1. PMID: 27134877; PMCID: PMC4843263.
C4d staining is classified into 4 categories with the following definitions:
Diffuse: >50% staining of Peritubular capillaries (PTC) positive.
Focal: Staining of at least 10 PTC and between 10 – 50%.
Focal minimal: Staining of at least 3 but less than 10 PTC.
Negative: Staining of less than three PTC or completely negative.
AMR- Associated risk with different C4d staining categories:
To diagnose AMR it’s not only dependant at C4d Staining, although patients with DSA who are C4d positive has lower graft survival than those who are C4d negative which means that C4d staining strength significance is different between the different categories.
C4d staining significance is also dependant on the technique used. As we discussed previously there are two techniques, IHC and IF. IHC is considered more specific while IF is considered more sensitive. For that an advice to use both techniques is valid.
C4d staining occurs after activation of the complement system and deposition of C4d product in PTC. That means the more staining we have the more complement system was activated, the more antibodies to react and the more associated histological damage.
Diffuse C4d positive staining in the PTC is associated with acute AMR with higher chance to have DSA at time of biopsy, and correlated with poor graft survival.
. References:
Christian A. Seemayer1 , Ariana Gaspert2 , Volker Nickeleit3 and Michael J. Mihatsch.C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques. Nephrol Dial Transplant (2007) 22: 568–576.doi:10.1093/ndt/gfl594.
Nadasdy GM, Bott C, Cowden D, Pelletier R, Ferguson R, NadasdyT. Comparative study for the detection of peritubular capillary C4ddeposition in human renal allografts using different methodologies.Human Pathol 2005; 36: 1178–1185
We can see C4d staining positive without histological abnormalities. That’s can happen in many situations such as:
ABO-incompatible transplantations and graft accommodation.
Systemic Auto-immune disease.
Pregnancy.
“smouldering” AMR rejection.
References:
Dickenmann M, Steiger J, Descoeudres B, Mihatsch M, Nickeleit V. The fate of C4d positive kidney allografts lacking histological signs of acute rejection. Clin Nephrol. 2006 Mar;65(3):173-9. doi: 10.5414/cnp65173. PMID: 16550748.
Cohen D, Colvin RB, Daha MR, et al. Pros and cons for C4d as a biomarker. Kidney Int. 2012;81(7):628-639. doi:10.1038/ki.2011.497
Tahani Ashmaig
3 years ago
▪︎C4d is a classical pathway complement degradation product that, when detected in peritubular capillaries, correlates with antibody-mediated rejection, and is associated with poor renal allograft outcome [1].
☆C4d staining:
________________
▪︎Is scored semiquantitatively in four categories [2]:
1. No C4d staining (0% of (peritubular) capillaries)
2. Minimal C4d staining (0–10% of (peritubular) capillaries)
3. Focal C4d staining (10–50% of (peritubular) capillaries)
4. Diffuse C4d staining (>50% of (peritubular) capillaries)
▪︎Immunofluorescence is a more sensitive method to detect C4d than immunohistochemistry.
▪︎Diffuse C4d staining in peritubular capillaries (PTCs) during an acute rejection episode (ARE) is the footprint of antibody-mediated rejection [3].
▪︎Although a diffuse C4d staining is defined as positive, the definition and clinical significance of ‘minimal’ and ‘focal’ C4d staining remain debated issues.
▪︎Most experts consider a focal staining pattern as a red flag, especially when detected on paraffin-embedded tissue or in the presence of donor-specific antibody and/or suspicious histopathological features.
☆Issues for clinicians working with C4d in daily practice [2]:
__________________
▪︎Focal staining :
1. In paraffin-embedded tissue combined with a positive test for donor-specific antibody (DSA) and histopathological features: Most laboratories consider this as ‘positive for antibody-mediated rejection (AMR)’.
2. In frozen tissue combined with a positive test for DSA and histopathological features: This is sometimes called a ‘probable AMR’.
☆How to proceed?
▪︎In case of diffuse C4d staining in peritubular capillaries and histological evidence for AMR: Most experts would advise to treat the patient for AMR.
▪︎In case of focal C4d staining and histological evidence for AMR: Check for other possible underlying diseases. If no other cause can be found: Treat the patient for AMR.
▪︎In case of focal or diffuse C4d staining and absence of histological changes the decision as to whether to treat for AMR is more uncertain. Treating or close follow-up are both suitable options.
▪︎C4d positivity in grafts without histological abnormalities can be seen in ABO-incompatible grafts. In case of no histopathology and no graft dysfunction: It is suggested to interpret this as ‘graft accommodation’.
_______________
Ref:
[1] Ibrahim Batal, et al “The significance of renal C4d staining in patients with BK viruria, viremia, and nephropathy”. Modern Pathology volume 22, pages1468–
[2] Danielle Cohen, Robert B. Colvin. “Pros and cons for C4d as a biomarker”
[3] Johanna M Botermans, Hanneke de Kort, […], and Marian C. van Groningen
“C4d Staining In Renal Allograft Biopsies with Early Acute Rejection and Subsequent Clinical Outcome”. https://dx.doi.org/10.2215/CJN.07820910
diagnosis of ABMR according to Banff 2017 based on -pathological evidence of tissue injury includes > 1. Acute thrombotic microangiopathy (TMA) in the absence of any other cause was also reported as ABMR. 2. Glomerulitis (g>0) – Glomerulitis was defined as the percentage of glomeruli that revealed infiltration by leukocytes in the capillaries. Score g1, g2, and g3 were offered if 50% of glomeruli showed leukocytic infiltration respectively. 3. Peritubular capillaritis – The percentage of PTCs in the renal cortex that contained neutrophils or mononuclear cells was defined as peritubular capillaritis. A score of ptc1 was offered if 10 cells/PTC. -evidence of antigen antibodies interactions C4d scoring – Demonstration of PTC C4d deposition by immunohistochemistry was considered positive if grade was >C4d0. Score of C4d1, C4d2, and C4d3 was given if 1–9%, 10%–50%, and >50% PTCs were involved, respectively. endothelium gene transcripts related to ABMR -serological evidence of dsa or c4d there is retrospective observetional study of (404) biopsies revealed immunological injury. Of these, · 27.7% of the biopsies revealed pure ABMR (Group 1), · 60.6% revealed combined ABMR with TCR (Group 2) · 11.3% revealed pure TCR (Group 3). · The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%, of which C4d-negative rejections were 18.48% and 18.7% in Group 1 and Group 2 The mean SCr at the end of three years follow-up in patients with C4d-negative rejections was comparatively higher. C4d-negative ABMR, recently included in Banff’13, has a low incidence, usually presents early after transplantation but carries better outcome than C4d-positive ABMR. However, further long-term studies are still required for knowing the clinical course over years.
C4d can be detected in formalin-fixed and paraffine mbedded tissue sections employing a rabbit polyclonal antibody.
Scoring of the C4d expression in renal allograft biopsies is
(i) Diffuse expression: >50% of PTC positive.
(ii) Focal expression: staining of at least 10 PTC and <50%.
(iii) Focal minimal expression: staining of at least 3 but less than 10 PTC.
(iv) Negative: staining of less than three PTC or completely negative.
significance of C4D stainning,
diffuse C4d staining is associated with the highest risk of ABMR.
focal stainning is associated with DSA and ABMR.
focal minimal,some studies showed minimal risk of ambr with chroncity.
C4d negative staining in ptc if associated with evidence of high expression of (ENDATs), micro-vascular inflammation,& DSA is sufficient for diagnosis of AMR.
reference
1-Christian A. Seemayer1 , Ariana Gaspert2 , Volker Nickeleit3 and Michael J. Mihatsch.C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques.Nephrol Dial Transplant (2007) 22: 568–576.doi:10.1093/ndt/gfl594.
Last edited 3 years ago by MOHAMMED GAFAR medi913911@gmail.com
C4d deposition in peritubular capillaries can occur in one of the following:
1-Negative or minimal if there is a little or missing presence (0-10 per cent ptc). When conducted by immunohistochemistry,
2-Focal (>10-50 per cent PTC) results are considered positive.
3-Diffuse (more than 50% ptc) .
diffuse C4d staining detected by IF may be mistaken for focal deposition on IHC, which is more specific but less sensitive by about one grade level (diffuse C4d staining detected by IF may be mistaken for minimal by IHC, and focal staining detected by IHC may be mistaken for minimal by IF).
C4d-positive and C4d-negative ABMR shows similar frequencies of concurrent cell- mediated rejection and both can occur early or late posttransplant. Graft outcomes were com- paratively better in the cohort of patients who developed C4d-negative ABMR as compared to those who have C4d-positive ABMR.
C4d staining is classified into diffuse, focal, minimal and negative.
Scoring of C4d staining is based on the percentage of peritubular capillaries and vasa recta that has a linear, circumferential staining pattern
C4d0—No staining of PTC and medullary vasa recta (0%).
C4d1—Minimal C4d staining (>0 but <10% of PTC and medullary vasa recta).
C4d2—Focal C4d staining (10-50% of PTC and medullary vasa recta).
C4d3—Diffuse C4d staining (>50% of PTC and medullary vasa recta).
When deposition is diffuse mean there is strong correlation with anti-HLA Abs with poor graft outcome.
Reference
Roufosse, Candice MD, PhD1,2; Simmonds, Naomi MD3; Clahsen-van Groningen, Marian MD, PhD4; Haas, Mark MD, PhD5; Henriksen, Kammi J. MD6; Horsfield, Catherine MD3; Loupy, Alexandre MD7; Mengel, Michael MD8; Perkowska-Ptasińska, Agnieszka MD9; Rabant, Marion MD, PhD10; Racusen, Lorraine C. MD11; Solez, Kim MD8; Becker, Jan U. MD12 A 2018 Reference Guide to the Banff Classification of Renal Allograft Pathology, Transplantation: November 2018 – Volume 102 – Issue 11 – p 1795-1814 doi: 10.1097/TP.0000000000002366
Wael Jebur
3 years ago
C4d is the foot print of DSAs related AMR. It’s deposited and bound covalently to capillary endothelial basement membrane ,where DSAs -endothelial Antigen HLA and nonHLA ) interact.
Diffuse distribution is strongly associated with Capillaritis in the form of glomerulitis and peritubular capillaritis (g+ptc>2) referred to as Micro vascular inflimmation MVI,as per Banff classification.
Diffuse C4d staining is associated with high circulating DSAs titer .
It’s usually predisposed to, by preformed DSAs, those DSAs are Complement fixing and precipitate active AMR.
In the contrary AMR clinical phenotype dose not correlate with severity of underlying MVI.
Smoldering AMR:
In other cases C4d might be deposited focally , in association with mild MVI.
Focal C4d is detected in the following circumstances:
1) in asymptomatic patients ,on protocol biopsy
2) Normal allograft function.
3) No proteinuria.
4)Subclinical AMR.
4) It might be diffuse, but most commonly focal.
Etiology :
1) low circulating level of DSAs
2) Mostly non-complement fixing DSAs.
Significance and prognosis:
This focal C4d staining portend high risk for development of TG. As low grade MVI continued to trigger endothelial damage predisposing to TG and PTC multilayring.
TG is linked to C4d positivity.
It’s difficult and quite challenging to treate.
Negative C4d AMR:
In some cases of active and chronic active AMR, C4d staining is negative.
DSAs are positive.
Mechanism is Antibody mediated neutrophils and Natural killer cytotoxicity.
C4d is the foot print of DSAs related AMR. It’s deposited and bound covalently to capillary endothelial basement membrane ,where DSAs -endothelial Antigen HLA and nonHLA ) interact.
Diffuse distribution is strongly associated with Capillaritis in the form of glomerulitis and peritubular capillaritis (g+ptc>2) referred to as Micro vascular inflimmation MVI,as per Banff classification.
Diffuse C4d staining is associated with high circulating DSAs titer .
It’s usually predisposed to, by preformed DSAs, those DSAs are Complement fixing and precipitate active AMR.
In the contrary AMR clinical phenotype dose not correlate with severity of underlying MVI.
Smoldering AMR:
In other cases C4d might be deposited focally , in association with mild MVI.
Focal C4d is detected in the following circumstances:
1) in asymptomatic patients ,on protocol biopsy
2) Normal allograft function.
3) No proteinuria.
4)Subclinical AMR.
4) It might be diffuse, but most commonly focal.
Etiology :
1) low circulating level of DSAs
2) Mostly non-complement fixing DSAs.
Significance and prognosis:
This focal C4d staining portend high risk for development of TG. As low grade MVI continued to trigger endothelial damage predisposing to TG and PTC multilayring.
TG is linked to C4d positivity.
It’s difficult and quite challenging to treate.
Negative C4d AMR:
In some cases of active and chronic active AMR, C4d staining is negative.
DSAs are positive.
Mechanism is Antibody mediated neutrophils and Natural killer cytotoxicity.
C4d is the foot print of DSAs related AMR. It’s deposited and bound covalently to capillary endothelial basement membrane ,where DSAs -endothelial Antigen HLA and nonHLA ) interact.
Diffuse distribution is strongly associated with Capillaritis in the form of glomerulitis and peritubular capillaritis (g+ptc>2) referred to as Micro vascular inflimmation MVI,as per Banff classification.
Diffuse C4d staining is associated with high circulating DSAs titer .
It’s usually predisposed to, by preformed DSAs, those DSAs are Complement fixing and precipitate active AMR.
In the contrary AMR clinical phenotype dose not correlate with severity of underlying MVI.
Smoldering AMR:
In other cases C4d might be deposited focally , in association with mild MVI.
Focal C4d is detected in the following circumstances:
1) in asymptomatic patients ,on protocol biopsy
2) Normal allograft function.
3) No proteinuria.
4)Subclinical AMR.
4) It might be diffuse, but most commonly focal.
Etiology :
1) low circulating level of DSAs
2) Mostly non-complement fixing DSAs.
Significance and prognosis:
This focal C4d staining portend high risk for development of TG. As low grade MVI continued to trigger endothelial damage predisposing to TG and PTC multilayring.
TG is linked to C4d positivity.
It’s difficult and quite challenging to treate.
Negative C4d AMR:
In some cases of active and chronic active AMR, C4d staining is negative.
DSAs are positive.
Mechanism is Antibody mediated neutrophils and Natural killer cytotoxicity.
C4d is the foot print of DSAs related AMR. It’s deposited and bound covalently to capillary endothelial basement membrane ,where DSAs -endothelial Antigen HLA and nonHLA ) interact.
Diffuse distribution is strongly associated with Capillaritis in the form of glomerulitis and peritubular capillaritis (g+ptc>2) referred to as Micro vascular inflimmation MVI,as per Banff classification.
Diffuse C4d staining is associated with high circulating DSAs titer .
It’s usually predisposed to, by preformed DSAs, those DSAs are Complement fixing and precipitate active AMR.
In the contrary AMR clinical phenotype dose not correlate with severity of underlying MVI.
Smoldering AMR:
In other cases C4d might be deposited focally , in association with mild MVI.
Focal C4d is detected in the following circumstances:
1) in asymptomatic patients ,on protocol biopsy
2) Normal allograft function.
3) No proteinuria.
4)Subclinical AMR.
4) It might be diffuse, but most commonly focal.
Etiology :
1) low circulating level of DSAs
2) Mostly non-complement fixing DSAs.
Significance and prognosis:
This focal C4d staining portend high risk for development of TG. As low grade MVI continued to trigger endothelial damage predisposing to TG and PTC multilayring.
TG is linked to C4d positivity.
It’s difficult and quite challenging to treate.
Negative C4d AMR:
In some cases of active and chronic active AMR, C4d staining is negative.
DSAs are positive.
Mechanism is Antibody mediated neutrophils and Natural killer cytotoxicity.
Ban Mezher
3 years ago
Criteria for C4d staining is linear & circumferential deposition in at least 25% of cortex & medulla( excluding necrotic tissues). The distribution of the deposition can be classified into:
0- absent
1- minimal (<10%)
2- focal (10-50%)
3- diffuse (>50%)
Diffuse defined as positive for C4d deposition, focal & minimal staining remain uncertain issue. But most expert consider focal staining red flag especially if DSA was positive.
C4d staining in PTC is highly specific for presence of DSA, so when deposition is diffuse mean there is strong correlation with anti-HLA Abs with poor graft outcome.
References:
Carpio V., Rech C., Eickhoff E., Pegas K., Edelweiss M., and et al. Clinical and pathological correlation of C4d immunostaining and its influence on the outcome of kidney transplant recipients. Braz J Nephrol.2011;33(3).
Etta P. C4d Staining and Antibody-mediated Rejection in Renal Transplantation:Current Status. 2020;14(3):197-201.
According to Banff guidelines significant C4d deposition in PCT is a prerequisite for diagnosis of ABMR
C4d deposition patters
1- diffuse pattern if involved more than 50 % of PTC , commonly associated with DSA presence and increased risk for ABMR and poor graft survival
2- focal C4d deposition if involve (10-50%) of PTC : usally no increased risk of ABMR
3-Minimal: if less than deposition involve less than 10 %
4- c4d negative ABMR : no C4d deposition but associated with histological evidence of tissue damage and presence of DSA as in case of non complement fixing Ab mediated rejection or aoolantibody against FC receptors on NK cells
Worthington JE, McEwen A, McWilliam LJ, Picton ML, Martin S. Association between C4d staining in renal transplant biopsies, production of donor-specific HLA antibodies, and graft outcome. Transplantation. 2007;83(4):398-403. doi:10.1097/01.tp.0000251430.11723.b6
Ahmed Faisal
3 years ago
☆ C4d Stain Interpretation:
C4d0—No staining of PTC and medullary vasa recta (0%).
C4d1—Minimal C4d staining (>0 but 50% of PTC and medullary vasa recta).
C4d2—Focal C4d staining (10-50% of PTC and medullary vasa recta).
C4d3—Diffuse C4d staining (>50% of PTC and medullary vasa recta).
☆ Banff Lesion Score C4d :
The score evaluates the extent of staining for C4d on endothelial cells of PTCs and medullary vasa recta by IF on snap frozen sections of fresh tissue or IHC on formalin-fixated and paraffin-embedded tissue.
Scoring of C4d staining is based on the percentage of peritubular capillaries and vasa recta that has a linear, circumferential staining pattern.
The minimal sample for evaluation is 5 high-power fields of cortex and/or medulla without scarring or infarction. C4d must not be scored in areas of infarction.
In terms of extent of staining, with IF, Banff Lesion Score C4d ≥ 2 is considered positive and a criterion for antibody interaction with tissue and as equivalent to DSA, whereas with IHC, Banff Lesion Score C4d ≥ 1 is counted as positive already.
Diffuse C4d staining pattern is significantly associated with the presence of circulating donor-specific antibodies and higher risk of ABMR.
—————-
Reference
(1) Cornell LD (2021) Histopathologic Features of Antibody Mediated Rejection: The Banff Classification and Beyond. Front. Immunol. 12:718122. doi: 10.3389/fimmu.2021.718122
(2) Roufosse, C., Simmonds, N., Clahsen-van Groningen, M., Haas, M., Henriksen, K. J., Horsfield, C., Loupy, A., Mengel, M., Perkowska-Ptasińska, A., Rabant, M., Racusen, L. C., Solez, K., & Becker, J. U. (2018). A 2018 Reference Guide to the Banff Classification of Renal Allograft Pathology. Transplantation, 102(11), 1795–1814. https://doi.org/10.1097/TP.0000000000002366
Complement component 4 fragment d (C4d) is a degradation product of complement component C4, which can bind covalently to the endothelial cell surface.
Positive immunostaining indicates local complement activation and is scored with Banff Lesion Score C4d.
The clinical significance of these findings is different in grafts exposed to anti-blood group antibodies (ABO-incompatible allografts), in which isoagglutinin-mediated complement activation does not appear to be injurious to the graft and may represent accommodation.
However, when caused by donor-specific antibodies against HLA or other antigens, it may indicate AMR activity. Based on the high specificity of C4d positivity, it is considered equivalent to the serological proof of donor-specific antibody (DSA) for a diagnosis of AMR, although it is still recommended to test for DSA.
A, IHC staining
An example of C4d3, this image demonstrates linear and circumferential staining of endothelial cells in virtually all peritubular capillaries.
B, IF staining for C4d.
This image shows an example of a Banff Lesion Score of C4d3; using IF, a minimum score of C4d ≥ 2 is considered positive.
Mohamed Saad
3 years ago
C4d scoring –
Demonstration of PTC C4d deposition by immunohistochemistry was considered positive if grade was >C4d0.
Score of C4d1, C4d2, and C4d3 was given if 1–9% (minimal staining), 10%–50% (focal), and >50% PTCs were involved (diffuse), respectively.
C4dnegative ABMR was defined as biopsies with C4d <2 on immunofluorescence or C4d >0 on IHC, ptc> 0 and g >0, g+ptc ≥2, or g>0 or ptc >0 and acute TMA in the absence of any other cause of TMA(1).
C4d staining predict presence of DSA with sensitivity more than 95%..
In general C4d positive has a poor graft survival than C4d negative(2)
Detection of intragraft complement activation has strong independent value as an additional indicator of ABMR associated with adverse kidney transplant outcomes(2).
The worst 8-year death-censored graft survival was observed in patients scored as C4d3 ,followed by C4d2 , C4d1 , and C4d0 respectively (2)(figure2). References:
1-BANFF classification 2017.
2- Željko Kikić, Alexander Kainz et al, Capillary C4d and Kidney Allograft Outcome in Relation to Morphologic Lesions Suggestive of Antibody-Mediated Rejection, CJASN August 2015, 10 (8) 1435-1443; DOI: https://doi.org/10.2215/CJN.09901014.
Last edited 3 years ago by Mohamed Saad
saja Mohammed
3 years ago
C4D refer to complement split product of the classic complement pathway activation by antibodies antigen allo-immune response on the endothelial surfaces its essential marker for the diagnosis of the AMR since 1990s and IHC staining for C4Ddeposition in PTCs is one’s diagnostic criteria for AMR, Ca ABMR according to Banff histological diagnostic criteria scores since 2003, 2005 and update in 2017. with high specificity but restricted sensitivity in the era of C4D negative complement independent ABMR .
C4D staining:
-Glomerular deposition (mesangial and vascular pole) of C4D can be seen in normal kidney
-Diffuse staining defines by ≥ 50% of linear deposition of C4D in PTCS which is specific for the diagnosis of AMR.
-Focal staining defined by < 50% of C4D +ve in PTCS, when the biopsy performed in early stage of ABMR as the DSA complement activation proceed the C4D split product disposition , so still can be corelated with ABMR .
-Little or negative C4D staining in PTCS with typical histological evidence of ABMR and positive DSA can be seen in CD4 negative complement Independent ABMR, with or without DSA, usually late onset, more with Class 11 denovo DSA or non HLA antibodies , progressive but less aggressive course and poor response to anti-rejection therapy as its association with chronic transplant glomerulopathy and progressive graft loss, using molecular gene endothelial transcript ( ENDAT) which is molecular marker of endothelial cellular activation and injury can confirm the diagnosis
-C4D detection by either using immuno-histochemical staining (IHC) on formalin embedded paraffin sections with positive score >0, and ≥1% positive c4d staining of the PTC, its more specific but less sensitive compared to IF staining.
IF staining by using fresh frozen section with scoring of C4d is significant if 10% and above.
-C4d positive alone without evidence of endothelial injury is still of uncertain significance and it’s not enough to diagnosis AMR, like in case of ABOI transplant which indicate the graft accommodation.
-Molecular gene expression analysis in C4d+ve without evidence of allograft injury has the potential to identify patients at higher risk of developing ABMR (2).
Also the correlation of C4D staining with DSA positive assay for the diagnosis of ABMR can be challenged in case of low titer of DSA below the detection level or due to presence of non-HLA abs like ATIR antibodies , anti-endothelial ABs , which can be seen in aggressive type of ABMR with c4d +VE ,DSA -ve
References:
1- Haas M. The significance of C4d staining with minimal histologic abnormalities. Curr Opin Organ Transplant. 2010 Feb;15(1):21-7. doi: 10.1097/MOT.0b013e3283342ebd. PMID: 19907328.
2- Dominy KM, Willicombe M, Al Johani T, Beckwith H, Goodall D, Brookes P, Cook HT, Cairns T, McLean A, Roufosse C. Molecular Assessment of C4d-Positive Renal Transplant Biopsies Without Evidence of Rejection. Kidney Int Rep. 2018 Sep 18;4(1):148-158. doi: 10.1016/j.ekir.2018.09.005. PMID: 30596178; PMCID: PMC6308373.
3-kidney transplant in adult: clinical features and diagnosis of acute renal allograft rejection: up to date 2022.
C4d is a footprint for ABMR. It is found in peritubular capillaries and the vasa recta. It could be detected by either immunofluorescence or immunohistochemistry. Staining could be scored according to staining intensity to
Diffuse (score 3): more than 50% (linear or finely granular)
Focal (score 2): 10-5%
Minimal (score 1): < 10% and more than 0
Negative no staining
Diffuse staining is well correlated with the presence of DSA in the serum and with poorer graft outcomes as it means more complement activation. But in the case of ABOi transplant diffuse staining without evidence of tissue injury could be found with accommodation rather than rejection
-Minimal staining is considered significant by IF.
No. The risk of AMR in term of pattern of C4d staining is variable.
It is common to have histologic evidence of ABMR and a positive DSA but negative C4d staining in the peritubular capillaries. Such condition should treated using the same approach as that for patients with C4d-positive ABMR.
Reference
1)Presentation and Outcomes of C4d-Negative Antibody-Mediated Rejection After Kidney Transplantation. Orandi BJ. Am J Transplant. 2016 Jan;16(1):213-20.
C4d stain either minimal , focal , diffuse or negative
Minimal 0-10 % of PTC less significant
Focal 10-50 % OF PTC considered significant
Diffuse > 50 % OF PTC high significant
C4d staining is divided into 4 categories:
0: nil
1: minimal : staining >0 , <10% of PTCs indicates : less significant risk of ABMR
2: focal 10%-50% of PTCs indicate : significant risk of ABMR
3: diffuse= >50% of PTCs indicates : Higher significant risk of ABMR
Positive C4d depends on the used method for staining C4d.so:
Positive C4d staining : IF PTCs C4d staining scores equal or > 10%. or ;if in IHC PTCs C4d scores > 0.
Almost 90% of positive C4d are associated with presence of DSA.
C4d +ve but with negative DSA negative rejection can be seen in the following situations: ABO incompatible transplantation, auto-immune disease, and pregnancy.
C4d -ve/ positive DSA can be seen in the following conditions: Technical issues,non-HLA antibodies.
C4d staining is classified into diffuse, focal, minimal and negative.
· Are they all associated with the same risk of AMR?
· Substantiate your answer?
o The diagnosis of AMR requires the simultaneous presence of DSA, histo-pathological findings and the linear deposition of C4d in PTC. Additionally, there is a strong relationship between the presence of DSA and C4d staining.
o PTCs linear C4d staining either by immunofluorescence on frozen sections or immunohistochemistry on formalin-fixed, paraffin-embedded tissue can be scored as:
0: No staining
1: Minimal staining which is >0 but <10% of PTCs
2: Focal staining of 10%-50% of PTCs
3: diffuse staining of >50% of PTCs
o Immunofluorescence scores ≥2 are considered positive, whereas; Immunohistochemistry scores >0 are considered positive
o C4d is the immunological foot print for ABMR and complement activation, the greater the complement activation , the higher the risk for acute ABMR.SO,
o C4d can be negative in presence of DSA, this can occur in:
-Technical issues related to the type of fixative used
-Complement independent AMR
– autoantibodies to AT1R
-FcRIIA (Fc receptor on NK cells)
o Also, We can have C4d positive in the absence of rejection:
– graft accommodation in ABO-incompatible transplantations
– Pregnancy.
– Auto-immune diseases
References
1.Herzenberg A.M. Gill J.S. Djurdjev O. et al.C4d deposition in acute rejection: an independent long-term prognostic factor. J Am Soc Nephrol. 2002; 13: 234-241
2. Batal I, Girnita A, Zeevi A, Saab BA, Stockhausen S, Shapiro R, Basu A, Tan H, Morgan C, Randhawa P. Clinical significance of the distribution of C4d deposits in different anatomic compartments of the allograft kidney. Modern Pathology. 2008 Dec;21(12):1490-8.
C4d staing is scored as the following:
C4d0 Negative 0%
C4d1 Minimal 1<10%
C4d2 focal 10-50%
C4d3 diffuse > 50%
Diffuse PTC C4d staining may occur despite the absence of histologic findings of injury in recipients of ABO-incompatible kidney transplants.
on the other hand , Some recipients may have positive DSA and biopsy evidence of ABMR still lack detectable C4d staining.
PTCs C4d staining is divided into 4 categories:
0: nil
1: minimal=staining in >0 but <10% of PTCs.–> less significant risk of ABMR
2: focal=10%-50% of PTCs.->significant risk of ABMR
3: diffuse= >50% of PTCs.->Higher significant risk of ABMR
Positive C4d depends on the used method for staining C4d.
Positive C4d staining is when IF PTCs C4d staining scores equal or above 10%.
Positive C4d staining is when IHC PTCs C4d scores more than 0.
Almost 90% of positive C4d are associated with presence of DSA.
C4d +ve/ -ve DSA/ negative rejection can be seen in the following situations: ABO incompatible transplantation, auto-immune disease, and pregnancy.
C4d -ve/+ve DSA can be seen in the following conditions:
1-Technical issues, type of staining method.
2-some DSAs have poor complement fixation.
3-non-HLA antibodies like AT1R.
4-Complement independent ABMR.
Linear C4d in PTC either detected by IF (positive if >2) or IHC on frozen paraffin sections (positive if >0).
0: nil
1: (minimal, staining in >0 but 50% of PTCs)
2: (focal, 10%-50% of PTCs)
3: (diffuse, >50% of PTCs)
Diffuse C4d staining is considered significant clinically, and correlates well with DSA presence.
Focal staining is considered significant.
Minimal staining on IHC is considered significant except in IF
C4d negativity in presence of DSA is uncommon, but can be seen due to:
*Technical issues: type of fixative used, different methods of C4d detection.
* Poor complement fixation by some DSAs
* Complement independent AMR
* autoantibodies to AT1R
* FcRIIA (Fc receptor on NK cells)
In a case study: sensitivity of IHC in detecting acute AMR was only 55% and was 23.5% for chronic AMR.
Are they all associated with the same risk of AMR?
NO ACCORDING TO CLASSIFICATION OF C4D STAINING BY Frozen and paraffin section stained by immunoflorescence and immunohistochemistry SO THE GREATER THE PRESCENCE OF AFFECTION THE WORSET THE CONDTION AMR.
0: (none)
1: (minimal, staining in >0 but <10% of PTCs)
2: (focal, 10%-50% of PTCs)
3: (diffuse, >50% of PTCs)
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 is scored as:
0: (none)
1: (minimal, staining in >0 but <10% of PTCs)
2: (focal, 10%-50% of PTCs)
3: (diffuse, >50% of PTCs)
Immunofluorescence on frozen sections scores ≥2 are considered positive;
Immunohistochemistry on paraffin sections, all scores >0 are considered positive.
There is a strong relationship between the presence of DSA and C4d staining.
Diffuse C4d staining is considered significant clinically and correlates well with DSA presence. Focal staining is also considered significant. Minimal staining on IHC is considered significant, although on IF (as it is very sensitive) it is not considered sensitive.
C4d negativity in presence of DSA is uncommon but can be seen due to:
1) Technical issues: type of fixative used, different methods of C4d detection
2) Poor complement fixation by some DSAs
3) Complement independent AMR
4) AMR due to autoantibodies to AT1R
5) Fc receptor on NK cells
We can have C4d positive WITHOUT rejection in these situations:
C4d linear staining in PTCs or medullary vasa recta by immunofluorescence on frozen sections of fresh tissue or immunohistochemistry on formalin-fixed, paraffin-embedded tissue is 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 immunohistochemistry on paraffin sections all scores >0 are considered positive.
Studies revealed relation between presence of DSA and C4d staining. Sensitivity and specificity of C4d positive staining in a patient with DSA in acute AMR is very high ;more than 90%.
Diffuse C4d staining is clinically significant , and correlates well with DSA . Focal staining is also significant. Also, minimal staining on IHC is considered significant.
C4d negativity in presence of DSA is uncommon, but can be seen in case of :
– Technical issues: type of fixative used, and different methods of C4d detection
– Poor complement fixation by some DSAs
– Complement independent AMR
– AMR due to autoantibodies to AT1R
– Due to Fc RIIA ;Fc receptor on NK cells
Although C4d is a surrogate marker for DSAs, it has low sensitivity in AMR in late graft biopsies ;chronic AMR. It was found that sensitivity of IHC in detecting acute AMR is only 55% and 23.5% for chronic AMR. .
Are they all associated with the same risk of AMR:
yes.
C4d staining pattern during an early ARE does not predict renal allograft function or
survival over time.
Ref:
Johanna M.Botermans,et al.C4d Staining In Renal Allograft Biopsies with Early Acute
Rejection and Subsequent Clinical Outcome.Clin J Am Soc Nephrol. 2011 May; 6(5):
1207–1213.
No, there´s difference in the risk. The greater the presence of complement, the greater the risk of AMR.
When assessing the relevance of C4d to allograft failure and accounting for microcirculatory inflamation as a confounder, diffuse C4d was significantly associated with allograft loss, whereas focal C4d was not. Finally, Verghese et al. C4d, did not significantly alter the risk for allograft loss.
REFERENCE:
– A systematic review of the role of C4d in the diagnosis of acute antibody-mediated rejection. Kidney International (2015) 87, 182–194; doi:10.1038/ki.2014.166;
C4d is the immunologic footprint of complement activation and AMR. Staining for C4d is detected by two methods: IHC and IF. If C4d staining in IHC involves more than 10% of PTCs, it is considered positive. But IF is reported positive only if it is diffuse which means involving more than 50% of PTCs. Focal involvement means less than 50% involvement of PTCs by IF C4d staining. C4d is 95% sensitive and 96% specific for DSA. In patients with DSA, positive C4d staining was associated with worse graft function and survival. So, diffuse C4d is a marker for remarkable injury. Some patients have histologic evidence of ABMR and are DSA positive, but are C4d negative in PTCs. In TG only 36% were C4d +, but 73% were DSA positive. Complement independent mechanisms may be involved in these cases.
C4d negative ABMR should be treated as ABMR, otherwise they progress to TG and graft loss. But if treated may have better outcome. In addition, ABO incompatible TX, pregnancy and autoimmune diseases will have positive C4d.
# Peritubular capillary staining was classified as #Diffuse when staining was present in more than half of the microscopic fields examined.
# Staining was referred to as focal when present in 10–50%
# Minimal when 50%, 10–50%, or <10% of the microscopic fields showed the presence of tubular basement membrane C4d staining, respectively. The absence of detectable stain was regarded as negative. To facilitate statistical analysis, C4d staining in the peritubular capillaries and tubular basement membranes was also assigned a numerical score of 0 (negative), 1 (minimal), 2 (focal), or 3 (diffuse). Bowman's capsule staining was qualitatively recorded as present or absent.
Ahuja M, Cohen EP, Dayer AM, et al. Polyoma virus infection after renal transplantation. Use of immunostaining as a guide to diagnosis. Transplantation 2001;71:896–899.
C4D attaining is classified into diffuse, focal, minimal and negative
The diagnosis of acute AMR requires three components: histologic evidence (glomerulitis, peritubular capillaritis, intimal or transmural/necrotizing arteritis, or acute tubular injury without other apparent cause); evidence of recent antibody interaction with graft endothelium (C4d staining in at least 10% of peritubular capillaries by immunofluorescence on a frozen section or any peritubular capillary C4d staining by immunohistochemistry), and presence of DSA.
The C4d score varied from 0 to 3 (negative, minimal, focal and diffuse) depending on the percentage of PTC having a linear staining pattern: no staining = score 0, <10%= score 1 (minimal), 10–50%= score 2 (focal) and >50%= score 3 (diffuse).
Microvascular inflammation and injury are not specific to acute AMR. Thus, staining for C4d should be performed on all renal allograft biopsies and routine testing for DSA is also strongly advisable, especially in sensitized patients and whenever AMR is in the differential diagnosis. Although C4d may not be a sufficiently sensitive indicator when compared to microvascular inflammation and DSA class II, that, are more strong indicators of chronic graft outcome.
Negative, minimal and focal/diffuse C4d are associated with simultaneous grades of microcirculation injury and class II DSA. There is notable variation in the C4d status in the first year post-transplant suggesting that ABMR may be a subclinical process, fluctuating in parallel with fluctuations of C4d status.
The correlation between the intensity of fibrosis and deposition of C4d was positive.
There was positivity for IgA, IgM and C3 in groups C4d0, 1, 2 and 3, for IgG and fibrinogen in groups C4d2 and 3, and for C1q in groups C4d1, 2 and 3. C4d3 group showed a higher percentage of fibrosis, with elevated serum creatinine levels, and a positive correlation between fibrosis and intensity of C4d expression.
Linear C4d staining of peritubular capillaries in the absence of evidence of acute tissue injury can be seen in accommodation, which is common in blood group ABO-mismatched transplants.
Granular C4d staining of peritubular capillaries can be seen in lupus nephritis.
(1–4)
1. Collins AB, Schneeberger EE, Pascual MA, Saidman SL, Williams WW, Tolkoff-rubin N, et al. Complement Activation in Acute Humoral Renal Allograft Rejection : Diagnostic Significance of C4d Deposits in Peritubular Capillaries. 1999;(11):2208–14.
2. Seemayer CA, Gaspert A, Nickeleit V, Mihatsch MJ. C4d staining of renal allograft biopsies : a comparative analysis of different staining techniques. 2007;(December 2006):568–76.
3. Corrêa RRM, Machado JR, da Silva MV, Helmo FR, Guimarães CSO, Rocha LP, et al. The importance of C4d in biopsies of kidney transplant recipients. Clin Dev Immunol [Internet]. 2013/07/09. 2013;2013:678180. Available from: https://pubmed.ncbi.nlm.nih.gov/23935649
4. Loupy A, Hill GS, Suberbielle C, Charron D, Anglicheau D, Zuber J, et al. Significance of C4d Banff scores in early protocol biopsies of kidney transplant recipients with preformed donor-specific antibodies (DSA). Am J Transplant Off J Am Soc Transplant Am Soc Transpl Surg. 2011 Jan;11(1):56–65.
– C4d0 No staining of PTCs (0%)
– C4d1 Minimal C4d staining (>0 but<10 % of PTCs) •
– C4d 2 Focal C4d staining (10–50% of PTCs)
– C4d3 Diffuse C4d staining (>50% of PTCs)
Diffuse C4d staining in peritubular capillaries (PTCs) during an acute rejection episode (ARE) is the footprint of antibody-mediated rejection. diffuse C4d+ staining during acute rejection is regarded as an inferior prognostic sign. patients with ABMR C4d⁺ had the worst graft survival over 3 years (p = 0.034).
Conclusions: This study confirmed that C4d staining in kidney graft biopsies is a clinically useful marker of ABMR, with well-defined clinical and pathological correlations. The impact of C4d deposition in other histologic diagnoses deserves further investigation.
Ref
Clinical and pathological correlations of C4d immunostaining and its influence on the outcome of kidney transplant recipients.
Carpio VN, Rech C, Eickhoff EI, Pegas KL, Edelweiss MI, Gonçalves LF, Manfro RC, Veronese FV.J Bras Nefrol. 2011 Jul-Sep;33(3):329-37. doi: 10.1590/s0101-28002011000300009.PMID: 22042350
C4d staining according to the % of PTC involved :
diffuse c4d positivity has the highest correlation with DSA and incidence of ABMR .
non complement fixing Ab
aggressive anti rejection Rx before biopsy
technical error)
( here there should be linear ( not circumfencial ) C4d staining , no histological features of A or C ABMR , No molecular evidence ( ENDAT ) of ABMR , NO A or C TCMR changes )
Are they all associated with the same risk of AMR?
diffuse c4d staining in PTC during acute rejection episode is the footprint of ABMR and also poor prognostic sign,
c4d staining in PTC was evaluated according to the area percentage of positive staining in the renal cortex there are three c4d staining group are made:
1- diffuse positive >50% of PTCs staining.
2-focal 10% to 50% of PTCs
3-negative<10% of PTCs.
staining was performed by both IF and IHC
c4d negative ABMR recently included in banff13 has low incidence,usually present early after transplantation but carries better out come than c4d positive ABMR,,
in diffuse c4d positive is associated higher risk for ABMR than focal or minimal(c4d .
negative), that mean not same risk of ABMR,
although both they have same histologist and serological requirement
support that c4d negative has risk of ABMR is present of chronic ABMR c4d negative histologist finding many studies is more prominent so that mean c4d negative may have same risk of ABMR c4d positive but is over looked.
thus,it appears necessary that diagnostic criteria for ABMR be revised to include c4 d negative lesion
References:
1. Terasaki PI, Cai J. Human leukocyte antigen antibodies and chronic rejection: from association to causation. Transplantation 2008; 86:377– 383.
2. Colvin RB. Antibody-mediated renal allograft rejection: diagnosis and pathogenesis. J Am Soc Nephrol 2007; 18:1046–1056.
Staining of C4d either by IF of frozen sections or IHC of paraffin-embedded sections
classified into :
all sections. C4d staining in PTCs was evaluated according to the area percentage of positive staining in the renal cortex as described in the Banff criteria. Three C4d staining groups were made:
Negative no staining
minimal (<10% of PTCs)
focal (10% to 50% of PTCs), and
diffuse positive (>50% of PTCs) staining
Negative C4d :
C4d negative ABMR better outcome but need aggressive treatment like positive c4d ABMR
The state where complement 4d is negative
The following conditions are showing C4d negativity
a. T‑cell‑mediated rejection (TCMR)
b. technical errors of fixation
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
Focal staining :
it may represent an evolving AMR. However, a focal staining pattern is frequently observed during the resolution of AMR episode
diffuse positive (>50% of PTCs) staining :
Indicate the presence of Alloantibodies directed against HLA antigen with complement-dependent pathway
With more prevalent in :
a. Acute AMR
b. Chronic antibody‑mediated rejection
c. A, B, O blood group ABO-incompatible grafts
d. Accommodation.
However Botermans et al retrospective study showed that C4d staining is not related to clinical outcomes in this large cohort of first histologically proven early (<6 months),
Reference :
The Relevance of Complement C4d Staining in Renal Allograft
Biopsies
Anju Khairwa*
C4d Staining In Renal Allograft Biopsies with Early Acute Rejection and Subsequent Clinical Outcome
Johanna M Botermans,* Hanneke de Kort,* Michael Eikmans,† Klaas Koop,* Hans J. Baelde,* Marko J.K. Mallat,‡ Kim Zuidwijk,‡ Cees van Kooten,‡ Emile de Heer,* Natascha N.T. Goemaere,§ Frans H.J. Claas,† Jan A. Bruijn,* Johan W de Fijter,‡ Ingeborg M. Bajema,* and Marian C. van Groningenfile:///C:/Users/new/AppData/Local/Temp/msohtmlclip1/03/clip_image001.gif
Acute Antibody-Mediated Rejection
Ivy A. Rosales, Robert B. Colvin, in Kidney Transplantation, Bioengineering and Regeneration
C4d is a degradation product of the classic complement pathway. IHC is less sensitive than immunofluorescent detection methods C4d scoring
0: (none)
1: (minimal, staining in >0 but <10% of PTCs)
2: (focal, 10%-50% of PTCs)
3: (diffuse, >50% of PTCs)
Presence of positive C4d associated with presence of DSA and most probably AMR . We can have +ve C4d in autoimmune disease ,pegnancy and ABO incompatible transplantation which consider as false positive
Negative C4d cannot rule out AMR it can be du to :
a- antibodies didn’t fix the complement
b- Fc -portion in NK cell
c- AMR du to auto-antibodies
d- Technical error
desensitized patients with AMR have similar outcomes regardless of C4d staining .
C4d is complement degradation product of C4b which is formed in the classical pathway. It has sulfahydryl groups which have strong covalent binding into the endothelium of the vasa recta and peri tubular capillaries. C4d is considered the foot print of ABMR representing deposition after HLA antigen and anti HLA antibody.
C4d staining can be done in renal allograft biopsies by IF (immunofluorescence) by using either mono clonal or polyclonal antibodies or by IHC (immunohistochemistry) from formalin embedded tissues. Banff Classification recognized C4d staining as a diagnostic marker of ABMR in 2003. In the 2007 Banff classification C4 d staining was scored and listed as follows
0 – C4d negative in PTC and medullary vasa recta
1 – C4d Positive >0 – <10% in PTC and medullary vasa recta
2 – C4d Positive – Focal – >10% in PTC and medullary vasa recta
3 – C4d Positive – Diffuse >50% in PTC and medullary vasa recta
2013 Banff conference has recommended that atleast 10 percent of C4d staining is recommended to define ABMR.
Diffuse C4d positivity is associated with higher levels of DSA and more severe ABMR than focal
C4d negative ABMR is associated with low levels of DSA which are usually class II DSA and are associated with late onset ABMR
C4d negative ABMR is also seen with non HLA mediated ABMR like anti endothelin or ATR1 receptor antibody mediated ABMR, FC receptor NK cell mediated ABMR, non complement binding DSA, low level of complement binding DSA, technique failure (improper methods)
C4d has been associated with glomerular deposits in diseases like Lupus nephritis, membranous nephropathy, minimal change disease. They signify classical pathway activation of complement
C4d, a complement split product, is regarded as a footprint of AMR. Peritubular capillary C4d staining was classified as diffuse when staining was present in more than half of the microscopic fields examined. Staining was referred to as focal or minimal when present in 10–50% or <10% of the microscopic fields, respectively. The absence of detectable staining was regarded as negative.
Botermans JM et al, found Sensitivity (95%) and specificity (96%) of diffuse C4d staining in PTCs for the presence of DSAs is high. Similarly, other studies have found a strong relation between diffuse C4d staining of PTCs and the presence of DSAs. However, he found no difference in the occurrence of steroid resistance or graft survival for patients who in retrospect showed a C4d+ or a C4d− graft rejections and he concluded that the C4d staining pattern during an early graft rejections does not predict renal allograft function or survival over time.
On the other hand, Batal I et al, found that diffuse C4d was associated with an inferior outcome even in the absence of demonstrable donor-specific antibodies.
However, Rosales IA et al, in a study describing serial allograft biopsies of pre-sensitized patients, found that the C4d was initially negative and later was noted to be positive. Similarly, others found that Antibody levels and C4d deposition can fluctuate with time and may be a less active process at the time of biopsy.
References:
1. Botermans JM, de Kort H, Eikmans M, Koop K, Baelde HJ, Mallat MJ, Zuidwijk K, van Kooten C, de Heer E, Goemaere NN, Claas FH. C4d staining in renal allograft biopsies with early acute rejection and subsequent clinical outcome. Clinical Journal of the American Society of Nephrology. 2011 May 1;6(5):1207-13.
2. Rosales IA, Colvin RB. Acute Antibody-Mediated Rejection. InKidney Transplantation, Bioengineering and Regeneration 2017 Jan 1 (pp. 475-487). Academic Press.
3. Colvin RB, Chang A. Diagnostic pathology: kidney diseases. Farris AB, editor. Amsterdam: Elsevier; 2016.
4. Batal I, Girnita A, Zeevi A, Saab BA, Stockhausen S, Shapiro R, Basu A, Tan H, Morgan C, Randhawa P. Clinical significance of the distribution of C4d deposits in different anatomic compartments of the allograft kidney. Modern Pathology. 2008 Dec;21(12):1490-8.
Diffuse C4d staining in peritubular capillaries (PTCs) during an acute rejection episode (ARE) is the footprint of antibody-mediated rejection. In current clinical practice, diffuse C4d+ staining during acute rejection is regarded as an inferior prognostic sign.
C4D is specific but not sensitive marker of ABMR
C4D negative is less common than C4D + cases
C4d+ve AMR: more often seen in patients with DSA against HLA class I, or both class I and II.
C4D positive ABMR occurs earlier and more severe than C4D -ve
Banff recommendations.
C4d
diffusely positive if ≥50% of PTCs
focal positive if <50%
negative if very little (<10%) or no staining was seen in accordance
C4d-negative antibody-mediated rejection in renal allografts is Less severe than C4d-positive AMR (potential for causing transplant glomerulopathy, IF/TA, and graft loss)
But If not treated, it will result in all of these. So , Aggressive treatment of c4d negative ABMR is recommended and anti Complement treatment is not effective
1-Johanna M Botermans, Hanneke de Kort, […], and Marian C. van Groningen. C4d Staining In Renal Allograft Biopsies with Early Acute Rejection and Subsequent Clinical Outcome. American Society of Nephrology.
2-Renal Allograft Rejection PART -III Tarek Medhat Abbas, MD Prof of Nephrology Mansoura University
In normal kidneys, C4d is detectable in the glomerular mesangium and at the vascular pole. Deposition of C4d in the PTCs has only been described in renal allografts and represents complement activity directed against donor antigen
Criteria for C4d staining is linear & circumferential deposition in at least 25% of cortex & medulla excluding necrotic tissues.
0- absent
1- minimal (50%)
C4d staining in PTC is highly specific for presence of DSA, so when deposition is diffuse mean there is strong correlation with anti-HLA Abs with poor graft outcome however C4d staining in ABO-i indicates ‘graft accommodation’ rather than AMR.
Introduction:
detection of C4d, a single complement degradation product that can independently predict unfavorable graft outcome. C4d participates in the classic pathway of complement activation. Activated C4 is cleaved to C4a and C4b. C4b binds to an amino or hydroxyl group and is then converted to C4d, which can covalently bind with peritubular capillary basement membrane.
Risk of AMR is :
Linear or finely granular peritubular capillary staining was classified as diffuse when it was seen in more than 50% of microscopic fields available for evaluation, focal when present in 10–50% of biopsied tissue, and negative if the staining did not even reach
that threshold.
diffuse pattern is highly correlated with circulating DSA and highest incidence of AMR followed by focal pattern.
In terms of extent of staining, with IF, Banff Lesion Score C4d ≥ 2 is considered positive and a criterion for antibody interaction with tissue and as equivalent to DSA.
C4d0—No staining of PTC and medullary vasa recta (0%).
C4d1—Minimal C4d staining (>0 but <10% of PTC and
medullary vasa recta).
C4d2—Focal C4d staining (10-50% of PTC and medullary
vasa recta).
C4d3—Diffuse C4d staining (>50% of PTC and medullary
vasa recta).
C4D negative samples could be false negative ( eg technical errors and may need another sample, or could be T cell mediated with scarce antibody level to be detected)
Reference Guide to the Banff Classification of Renal Allograft Pathology, Transplantation: November 2018 – Volume 102 – Issue 11 – p 1795-1814
C4d staining in PTCs either identified by IF on frozen sections of fresh tissue or IHC on, paraffin-section and is scored as:
0: (none)
1: (minimal, staining in >0 but <10% of PTCs)
2: (focal, 10%-50% of PTCs)
3: (diffuse, >50% of PTCs)
Immunofluorescence on frozen sections scores ≥2 are considered positive;
Immunohistochemistry on paraffin sections, all scores >0 are considered positive.
Diffuse C4d staining is considered significant clinically and correlates well with DSA presence. Focal staining is also considered significant. Minimal staining on IHC is considered significant, although on IF (as it is very sensitive) it is not considered sensitive.
There is a strong relationship between the presence of DSA and C4d staining.However, some exceptions exist:
· C4d negative AMR (+ve DSA) may be due to :
1. Technical errors
2. Different method of c4d detection (IF vs IHC).
3. Very low amount of c4d (difficult detection).
4. Poor complement fixation by DSA
5. Complement independent pathway AMR or antibody dependent cell mediated cytotoxicity.
6. Antibodies against Angiotensin II type 1 receptors
7. fc receptors of Nk may have a role c4d negative AMR.
8. Increased expression of endothelial transcripts causing endothelium injury.
9. Sure pure TCMR will have -ve c4d.however, mixed rejection may be either c4d +ve or -ve.
· C4d +ve staining without AMR (-ve DSA):
o Not every c4d +ve means AMR: (sometimes accommodation to antibodies occurs so no immunologically mediated damage occur).
o ABO incompatible transplantation will have +ve c4d.
o Immune mediated kidney disease as lupus nephritis or anti phospholipid syndrome (either recurrent in the graft or infrequently present in donor and not detected). However, mostly here the c4d deposits will be mesangial or glomerular (not in peritubular capillaries).
C4d staining is classified into diffuse, focal, minimal and negative
Are they all associated with the same risk of AMR?
Substantiate your answer?
C4d linear staining in pertitubular capillary vasa recta by IF on Frozen sections of fresh tissues or IHC on formalin fixed, paraffin -embedded tissues is scored according to:
1- minimal staining >0 but 10% but 50% staining
IF C4d2 and more considered positive but IHC more than 0 is considered positive
Some conditions associated with diffuse C4d positivity without ABMR:
1- ABOi- due to graft accommodation
2- Pregnancy
3- Autoimmune diseases such as SLE
ABMR is diagnosed by presence of DSA, histological features that suggestive and C4d in PTC
Diffuse C4d is strongly associated with capillarities and TG which are the features of ABMR
C4d positivity in lupus nephritis is a bit tricky, it can be ABMR which need to be considered
C4d negative
C4d negative could be due to complement independent form of ABMR. Study by loupy showed that C4d or capillaritis in protocol biopsy determines the progression to TG and capilaritis without C4d also had higher rate of TG progression.
C4d negative ABMR usually overlooked and never been treated and study by Haas and Mirocha showed that C4d negative ABMR had higher rate of progression compared to treated C4d positive ABMR
References
1.Herzenberg A.M. Gill J.S. Djurdjev O. et al.C4d deposition in acute rejection: an independent long-term prognostic factor.J Am Soc Nephrol. 2002; 13: 234-241
2.Loupy A. Hill G.S. Suberbielle C. et al.Significance of C4d Banff scores in early protocol biopsies of kidney transplant recipients with preformed donor-specific antibodies (DSA). Am J Transplant. 2011; 11: 56-65
3.Haas M. Mirocha J. Early ultrastructural changes in renal allografts: correlation with antibody-mediated rejection and transplant glomerulopathy Am J Transplant. 2011
C4d staining is Frozen and paraffin section stained by immunoflorescence and immunohistochemistry.
C4d staining is not diagnostic for AMR without histological feature of acute or chronic AMR.
There’s some conditions associated with C4d positive without histological feature like
– [ ] ABO incompatible
– [ ] Pregnancy
– [ ] Autoimmune disease
C4d staining is classified to
Diffuse > 50% peritubular capillarities and it’s associated with high level of DSA and it’s gold stander diagnosis of AMR and it’s footprints AMR.
Focal C4d staining is 10 to 50% peritubular capillarities
Minimal C4d staining less than 10% peritubular capillarities.
Negative C4d staining means no peritubular capillarities.
References:
Merk Hass, The significance of C4d staining with minimal histologic abnormalities; natural library of medicine curr open organ transplant: 2010 Feb.
Johanna M Botermans, et al; C4d Staining In Renal Allograft Biopsies with Early Acute Rejection and Subsequent Clinical Outcome: Clin J Am Soc Nephrol. 2011 May; 6(5): 1207–1213.
C4d scoring
Scoring of C4d staining is based on the percentage of
stained tissue on IF/IHC that has a linear, circumferential staining pattern in PTC : C4d0—No staining of PTC (0%).
C4d1—Minimal C4d staining0-10%
C4d2—Focal C4d staining 10-50%
C4d3—Diffuse C4d staining >50%
Presence of positive C4d associated with presence of DSA and most probably AMR except in autoimmune disease and transplant a cross ABO incompatible which consider here as false positive,on the other hand negative C4d cannot rule out AMR because there’s many antibodies didn’t fix the complement
As Cohen, Danielle et al. described in “Pros and cons for C4d as a biomarker.”
Immunofluorescence is a more sensitive d than immunohistochemistry but we need a fresh frozen section. In IHC the positivity carries more importance when detected.
Reference:
Kidney international vol. 81,7 (2012): 628-39. doi:10.1038/ki.2011.497
There is debate about the importance of cd negative AMR but we know that may be due to early stage. so In case of cd4 negative AMR as AMR may be present without effect on eGFR we need to correlate histologic findings with serial monitoring of GFR and DSA level
4. C4d staining is classified into diffuse, focal, minimal and negative.
Are they all associated with the same risk of AMR?
Substantiate your answer?
The diagnosis of AMR requires the simultaneous presence of DSA, characteristic histopathological findings & linear deposition of C4d in PTC.
Most centers include routine C4d staining in diagnostic pathology evaluation of all renal allograft biopsies.
More than 70–80% of ABO-i grafts showed diffuse C4d positivity.
In contrast to conventional transplants where a diffuse C4d stain is strongly associated with histological abnormalities such as capillaritis & TG, the ABO-I kidneys as a rule show diffuse C4d positivity without histological tissue injury.
C4d staining in ABO-i indicates ‘graft accommodation’ rather than AMR.
PTC C4d staining was tested in many forms of GN, where PTC C4d staining was virtually never observed.
The only exception was lupus nephritis, in which granular PTC staining has been rarely described, which should be kept in mind when a diagnosis of AMR in a transplanted SLE patient is considered.
C4d-negative AMR:
Molecular studies showed a complement-independent form of AMR or C4d-negative AMR, in which C4d is obviously not helpful as a diagnostic tool.
Loupy et al. showed that C4d or capillaritis in 3-month protocol biopsies were risk factors for later TG, & capillaritis was predictive even in the absence of C4d.
Haas & Mirocha investigated patients with DSAs who had a biopsy during the first 3 months posttransplant. Patients with a C4d-negative biopsy who were not treated for AMR had a higher rate of progression to TG than those who were treated for AMR post-biopsy.
References
1.Herzenberg A.M. Gill J.S. Djurdjev O. et al.C4d deposition in acute rejection: an independent long-term prognostic factor.J Am Soc Nephrol. 2002; 13: 234-241
2.Loupy A. Hill G.S. Suberbielle C. et al.Significance of C4d Banff scores in early protocol biopsies of kidney transplant recipients with preformed donor-specific antibodies (DSA). Am J Transplant. 2011; 11: 56-65
3.Haas M. Mirocha J. Early ultrastructural changes in renal allografts: correlation with antibody-mediated rejection and transplant glomerulopathy Am J Transplant. 2011
Very good
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 is scored as:
0: (none)
1: (minimal, staining in >0 but <10% of PTCs)
2: (focal, 10%-50% of PTCs)
3: (diffuse, >50% of PTCs)
Immunofluorescence on frozen sections scores ≥2 are considered positive;
Immunohistochemistry on paraffin sections, all scores >0 are considered positive.
There is a strong relationship between the presence of DSA and C4d staining.
Diffuse C4d staining is considered significant clinically and correlates well with DSA presence. Focal staining is also considered significant. Minimal staining on IHC is considered significant, although on IF (as it is very sensitive) it is not considered sensitive.
C4d negativity in presence of DSA is uncommon but can be seen due to:
1) Technical issues: type of fixative used, different methods of C4d detection
2) Poor complement fixation by some DSAs
3) Complement independent AMR
4) AMR due to autoantibodies to AT1R
5) Fc receptor on NK cells
We can have C4d positive WITHOUT rejection in these situations:
Excellent
Concise and clear
Diffuse C4d staining in peritubular capillaries (PTCs) is considered as footprint of antibody-mediated rejection ,but is regarded as an inferior prognostic sign as C4d staining is not related to clinical outcome in the histologically proven early Acute rejection.(1)
Focal PTC C4d+ staining detected via IP was shown to be associated with circulating DSA and AMR . Combined focal and diffuse PTC C4d+ staining by IP has a similar sensitivity when compared with the IF method . Isolated glomerular C4d+ staining was detected in lupus nephritis and other glomerular diseases of native kidney biopsies and in transplant glomerulopathy of allograft biopsies , but its association with DSA and AMR in kidney transplant recipients is unclear.
Although diffuse C4d+ staining of PTC in ABO-compatible kidney allografts is strongly associated with AMR, C4d+ staining of PTC is common in ABO-incompatible kidney transplant recipients and may be seen in up to 80% of protocol biopsies without any acute rejection findings , suggesting that the presence of C4d on endothelial cells might play a role in accommodation of the graft in ABO-incompatible kidney transplantation and is not associated with AMR. It was reported that biopsies with minimal PTC C4d staining demonstrate gene expression profiles and Banff injury scores similar to focal/diffuse PTC C4d+ biopsies, are associated with circulating DSAs, acute and chronic AMR , therefore are a valid marker for AMR; however, isolated glomerular C4d+ biopsies do not have the gene expression profiles or association with circulating DSAs as seen with all PTC C4d+ groups and are not a marker for AMR.(2)
Graft dysfunction with diffuse positivity for C4d staining require specific anti-humoral therapy. Cases presenting with minimal and focal positivity for C4d showed improved graft function with pulse dose steroids and change in immunosuppression. C4d detection is a useful adjunct in the diagnosis of humoral responses in renal transplant patients presenting with graft dysfunction and helps in guiding clinical decisions for achieving long-term graft survival.(3)
C4d-negative AMR described in a chronic, but not in acute settings; led to using endothelial transcripts combined with DSA as a marker for AMR. Biopsies with high expression of these endothelial transcripts in combination with circulating DSA showing concurrent histopathological lesions of AMR had poor outcomes. Patients with a C4d-negative biopsy who were not treated for AMR had a higher rate of progression to transplant glomerulopathy than those who were treated for AMR post-biopsy.(4)
Reference
1-Botermans J M et al. C4d Staining In Renal Allograft Biopsies with Early Acute Rejection and Subsequent Clinical Outcome.Clin J Am Soc Nephrol. 2011 May; 6(5): 1207–1213.
2-Hayde, Nicole ; Bao, Yi; Pullman, James; Ye, Bin ‘; Calder, Brent R.; Chung, Monica; Schwartz, Daniel; Alansari, Ahmed; de Boccardo, Graciela; Ling, Min; Akalin, Enver . The Clinical and Molecular Significance of C4d Staining Patterns in Renal Allografts, Transplantation Journal: February 27, 2013 – Volume 95 – Issue 4 – p 580-588
3-Guduru SL, Kuruvilla S, Abraham G, Mathew M, Saravanan S. Diagnostic efficacy of C4d immunostaining in the detection of the humoral component of renal allograft rejection and therapeutic implications. Indian J Pathol Microbiol 2009;52:345-8.
4-Cohen D etal . Pros and cons for C4d as a biomarker. Kidney Int. 2012 April ; 81(7): 628–639.
Very good knowledge but needs more organization
Scoring of the C4d expression in renal allograft biopsies
Biopsies were scored into four categories for the circumferential expression of C4d along PTC in the unscarred renal cortex:
1.Diffuse expression: >50% of PTC positive.
2. Focal expression: staining of at least 10 PTC and <50%.
3. Focal minimal expression: staining of at least 3 but less than 10 PTC.
4. Negative: staining of less than three PTC or completely negative.
-c4d positivity is correlated with ABMR, and even a focal c4d staining in the specimen is well-associated with active humoral rejection.
-Diffuse peritubular capillary C4d deposition in renal allograft biopsies is associated with donor-specific antibodies (DSA),ABMR and graft failure.
-focal C4d had similar impact on graft survival as diffuse pattern.
-focal minimal has minimal risk of ABMR.
-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
References:
1.Christian A. Seemayer, Ariana Gaspert et al. C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques. Nephrology Dialysis Transplantation, Volume 22, Issue 2, February 2007, Pages 568–576.
2-Diana Taheri, Ardeshir Talebi, Maryam Taghaodi et al. Pathological diagnosis of antibody-mediated rejection in renal allograft without c4d staining, how much reliable? ,Adv Biomed Res. 2012; 1: 40.
can you think of any variation in interpretation for ABOi transplants!
C4d staining in renal allograft biopsies can be –
1-Diffuse- >50 %of PTC are positive
2-Focal- 10-50% PTC are positive.
3- Focal minimal- Staining of at least 3 but <10 PTC
4-Negative- Completely negative or <3PTC
Significance of C4d staining
Focal staining can be associated with DSA and ABMR while diffuse staining carries high risk of ABMR due to tissue injury and microvascular inflammation. Focal minimal signifies chronic disease. In C4d negative cases the mechanism of injury is through NK cells and neutrophils and DSA are positive. Negative C4d staining if associated with High expression of ENDATs, DSA and microvascular inflammation are enough for AMR diagnosis.
Reference:
Johanna M Botermans et al. C4d Staining In Renal Allograft Biopsies with Early Acute Rejection and Subsequent Clinical Outcome. Clin J Am Soc Nephrol. 2011 May; 6(5): 1207–1213.
Decent arguments and as you implied C4d shows that the complement system is involved. A step further medicine is advancing, we know that complement targeted therapies shall be a primal future treatment option and a marker such as C4d will identify patients susceptible to those treatments
C4d staining read as-
1.No c4d staining-0% of ptc
2.minimal c4d-1-10% of ptc
3.focal c4d-10-50% of ptc
4.diffuse c4d->50% of ptc
These all grading are not associated with same risk of AMR.
C4d staining done by two methods-
A.IF study is more sensitive
B.IHC study is more specific
So,when IF study used focal and diffuse c4d staining marked as one of the character for ABMR, but when IHC study is used minimal, focal,diffuse c4d staining used as criteria in presence of histological injury and DSA.So the method of c4d staining defines the risk, but obviously in presence of other criteria like histological injury +presence of DSA.
The answer needs a more detailed explanation.
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 is scored as: (1)
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.
Various studies have shown that there is a relation between the presence of DSA and C4d staining. The sensitivity and specificity of C4d positive staining in a patient with DSA (in acute AMR) is very high (more than 90%). (2-6)
Diffuse C4d staining is considered significant clinically, and correlates well with DSA presence. Focal staining is also considered significant. Minimal staining on IHC is considered significant, although on IF (as it is very sensitive) it is not considered sensitive.
C4d negativity in presence of DSA is uncommon, but can be seen due to: (7)
1) Technical issues: type of fixative used, different methods of C4d detection
2) Poor complement fixation by some DSAs
3) Complement independent AMR
4) AMR due to autoantibodies to AT1R
5) Due to FcRIIA (Fc receptor on NK cells)
Although C4d is a surrogate marker for DSAs, it has low sensitivity in AMR in late graft biopsies (chronic AMR). (8) In fact, one study found that the sensitivity of IHC in detecting acute AMR was only 55% and was 23.5% for chronic AMR. (9)
Reference:
1) Loupy A, Haas M, Roufosse C, Naesens M, Adam B, Afrouzian M, Akalin E, Alachkar N, Bagnasco S, Becker JU, Cornell LD, Clahsen-van Groningen MC, Demetris AJ, Dragun D, Duong van Huyen JP, Farris AB, Fogo AB, Gibson IW, Glotz D, Gueguen J, Kikic Z, Kozakowski N, Kraus E, Lefaucheur C, Liapis H, Mannon RB, Montgomery RA, Nankivell BJ, Nickeleit V, Nickerson P, Rabant M, Racusen L, Randhawa P, Robin B, Rosales IA, Sapir-Pichhadze R, Schinstock CA, Seron D, Singh HK, Smith RN, Stegall MD, Zeevi A, Solez K, Colvin RB, Mengel M. The Banff 2019 Kidney Meeting Report (I): Updates on and clarification of criteria for T cell- and antibody-mediated rejection. Am J Transplant. 2020 Sep;20(9):2318-2331. doi: 10.1111/ajt.15898. Epub 2020 May 28. PMID: 32463180; PMCID: PMC7496245.
2) Troxell ML, Weintraub LA, Higgins JP, Kambham N. Comparison of C4d immunostaining methods in renal allograft biopsies. Clin J Am Soc Nephrol. 2006 May;1(3):583-91. doi: 10.2215/CJN.00900805. Epub 2006 Mar 29. PMID: 17699262.
3) Böhmig GA, Exner M, Habicht A, Schillinger M, Lang U, Kletzmayr J, Säemann MD, Hörl WH, Watschinger B, Regele H. Capillary C4d deposition in kidney allografts: a specific marker of alloantibody-dependent graft injury. J Am Soc Nephrol. 2002 Apr;13(4):1091-1099. doi: 10.1681/ASN.V1341091. PMID: 11912271.
4) Mauiyyedi S, Crespo M, Collins AB, Schneeberger EE, Pascual MA, Saidman SL, Tolkoff-Rubin NE, Williams WW, Delmonico FL, Cosimi AB, Colvin RB. Acute humoral rejection in kidney transplantation: II. Morphology, immunopathology, and pathologic classification. J Am Soc Nephrol. 2002 Mar;13(3):779-787. doi: 10.1681/ASN.V133779. PMID: 11856785.
5) Collins AB, Schneeberger EE, Pascual MA, Saidman SL, Williams WW, Tolkoff-Rubin N, Cosimi AB, Colvin RB. Complement activation in acute humoral renal allograft rejection: diagnostic significance of C4d deposits in peritubular capillaries. J Am Soc Nephrol. 1999 Oct;10(10):2208-14. doi: 10.1681/ASN.V10102208. PMID: 10505698.
6) Sapir-Pichhadze R, Curran SP, John R, Tricco AC, Uleryk E, Laupacis A, Tinckam K, Sis B, Beyene J, Logan AG, Kim SJ. A systematic review of the role of C4d in the diagnosis of acute antibody-mediated rejection. Kidney Int. 2015 Jan;87(1):182-94. doi: 10.1038/ki.2014.166. Epub 2014 May 14. PMID: 24827778.
7) Nigam LK, Vanikar AV, Kanodia KV, Patel RD, Suthar KS, Patel HV. C4d-negative antibody-mediated rejection: A pathologist’s perspective and clinical outcome. Saudi J Kidney Dis Transpl. 2018 Jan-Feb;29(1):39-49. doi: 10.4103/1319-2442.225206. PMID: 29456206.
8) Etta PK. C4d staining and antibody-mediated rejection in renal transplantation: Current status. Indian J Transplant 2020;14:197-201
9) Devadass CW, Vanikar AV, Nigam LK, Kanodia KV, Patel RD, Vinay KS, Patel HV. Evaluation of Renal Allograft Biopsies for Graft Dysfunction and Relevance of C4d Staining in Antibody Mediated Rejection. J Clin Diagn Res. 2016 Mar;10(3):EC11-5. doi: 10.7860/JCDR/2016/16339.7433. Epub 2016 Mar 1. PMID: 27134877; PMCID: PMC4843263.
excellent!
C4d staining is classified into 4 categories with the following definitions:
AMR- Associated risk with different C4d staining categories:
.
References:
The answer covers all points. My question is as follows: Can we see C4d positivity in grafts without histological abnormalities?
We can see C4d staining positive without histological abnormalities. That’s can happen in many situations such as:
References:
▪︎C4d is a classical pathway complement degradation product that, when detected in peritubular capillaries, correlates with antibody-mediated rejection, and is associated with poor renal allograft outcome [1].
☆C4d staining:
________________
▪︎Is scored semiquantitatively in four categories [2]:
1. No C4d staining (0% of (peritubular) capillaries)
2. Minimal C4d staining (0–10% of (peritubular) capillaries)
3. Focal C4d staining (10–50% of (peritubular) capillaries)
4. Diffuse C4d staining (>50% of (peritubular) capillaries)
▪︎Immunofluorescence is a more sensitive method to detect C4d than immunohistochemistry.
▪︎Diffuse C4d staining in peritubular capillaries (PTCs) during an acute rejection episode (ARE) is the footprint of antibody-mediated rejection [3].
▪︎Although a diffuse C4d staining is defined as positive, the definition and clinical significance of ‘minimal’ and ‘focal’ C4d staining remain debated issues.
▪︎Most experts consider a focal staining pattern as a red flag, especially when detected on paraffin-embedded tissue or in the presence of donor-specific antibody and/or suspicious histopathological features.
☆Issues for clinicians working with C4d in daily practice [2]:
__________________
▪︎Focal staining :
1. In paraffin-embedded tissue combined with a positive test for donor-specific antibody (DSA) and histopathological features: Most laboratories consider this as ‘positive for antibody-mediated rejection (AMR)’.
2. In frozen tissue combined with a positive test for DSA and histopathological features: This is sometimes called a ‘probable AMR’.
☆How to proceed?
▪︎In case of diffuse C4d staining in peritubular capillaries and histological evidence for AMR: Most experts would advise to treat the patient for AMR.
▪︎In case of focal C4d staining and histological evidence for AMR: Check for other possible underlying diseases. If no other cause can be found: Treat the patient for AMR.
▪︎In case of focal or diffuse C4d staining and absence of histological changes the decision as to whether to treat for AMR is more uncertain. Treating or close follow-up are both suitable options.
▪︎C4d positivity in grafts without histological abnormalities can be seen in ABO-incompatible grafts. In case of no histopathology and no graft dysfunction: It is suggested to interpret this as ‘graft accommodation’.
_______________
Ref:
[1] Ibrahim Batal, et al “The significance of renal C4d staining in patients with BK viruria, viremia, and nephropathy”. Modern Pathology volume 22, pages1468–
[2] Danielle Cohen, Robert B. Colvin. “Pros and cons for C4d as a biomarker”
[3] Johanna M Botermans, Hanneke de Kort, […], and Marian C. van Groningen
“C4d Staining In Renal Allograft Biopsies with Early Acute Rejection and Subsequent Clinical Outcome”.
https://dx.doi.org/10.2215/CJN.07820910
Very good
Needs to be reduced and organised
diagnosis of ABMR according to Banff 2017 based on
-pathological
evidence of tissue injury includes >
1. Acute thrombotic microangiopathy (TMA) in
the absence of any other cause was also reported as ABMR.
2. Glomerulitis (g>0) – Glomerulitis was
defined as the percentage of glomeruli that revealed infiltration by leukocytes
in the capillaries. Score g1, g2, and g3 were offered if 50% of glomeruli
showed leukocytic infiltration respectively.
3. Peritubular capillaritis – The percentage of PTCs in the renal cortex that contained neutrophils or mononuclear cells was defined as peritubular capillaritis. A score of ptc1 was offered if 10 cells/PTC.
-evidence of antigen antibodies interactions
C4d scoring – Demonstration of PTC C4d deposition by immunohistochemistry was considered positive if grade was >C4d0. Score of C4d1, C4d2, and C4d3 was given if
1–9%, 10%–50%, and >50% PTCs were involved, respectively.
endothelium gene transcripts related to ABMR
-serological evidence of dsa or c4d
there is retrospective observetional study of (404) biopsies revealed immunological
injury. Of these,
· 27.7% of the biopsies revealed pure ABMR (Group 1),
· 60.6% revealed combined ABMR with TCR (Group 2)
· 11.3% revealed pure TCR (Group 3).
· The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%, of which
C4d-negative rejections were 18.48% and 18.7% in Group 1 and Group 2
The mean SCr at the end of three years follow-up in patients with C4d-negative rejections was comparatively higher. C4d-negative ABMR, recently included in Banff’13, has a low incidence, usually presents early after transplantation but carries
better outcome than C4d-positive ABMR. However, further long-term studies are
still required for knowing the clinical course over years.
Reference
Saudi J Kidney Dis Transpl 2018;29(1):39-49 © 2018 Saudi Center for Organ Transplantation
Very good
Scoring of the C4d expression in renal allograft biopsies is
(i) Diffuse expression: >50% of PTC positive.
(ii) Focal expression: staining of at least 10 PTC and <50%.
(iii) Focal minimal expression: staining of at least 3 but less than 10 PTC.
(iv) Negative: staining of less than three PTC or completely negative.
significance of C4D stainning,
reference
1-Christian A. Seemayer1 , Ariana Gaspert2 , Volker Nickeleit3 and Michael J. Mihatsch.C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques.Nephrol Dial Transplant (2007) 22: 568–576.doi:10.1093/ndt/gfl594.
Excellent
thanks prof
C4d deposition in peritubular capillaries can occur in one of the following:
1-Negative or minimal if there is a little or missing presence (0-10 per cent ptc). When conducted by immunohistochemistry,
2-Focal (>10-50 per cent PTC) results are considered positive.
3-Diffuse (more than 50% ptc) .
diffuse C4d staining detected by IF may be mistaken for focal deposition on IHC, which is more specific but less sensitive by about one grade level (diffuse C4d staining detected by IF may be mistaken for minimal by IHC, and focal staining detected by IHC may be mistaken for minimal by IF).
C4d-positive and C4d-negative ABMR shows similar frequencies of concurrent cell- mediated rejection and both can occur early or late posttransplant. Graft outcomes were com- paratively better in the cohort of patients who developed C4d-negative ABMR as compared to those who have C4d-positive ABMR.
C4d-Negative Antibody-Mediated Rejection: A Pathologist’s Perspective and Clinical Outcome .Saudi J Kidney Dis Transpl 2018;29(1):39-49 © 2018 Saudi Center for Organ Transplantation
Assignment IV
C4d staining is classified into diffuse, focal, minimal and negative.
Scoring of C4d staining is based on the percentage of peritubular capillaries and vasa recta that has a linear, circumferential staining pattern
C4d0—No staining of PTC and medullary vasa recta (0%).
C4d1—Minimal C4d staining (>0 but <10% of PTC and medullary vasa recta).
C4d2—Focal C4d staining (10-50% of PTC and medullary vasa recta).
C4d3—Diffuse C4d staining (>50% of PTC and medullary vasa recta).
When deposition is diffuse mean there is strong correlation with anti-HLA Abs with poor graft outcome.
Reference
Roufosse, Candice MD, PhD1,2; Simmonds, Naomi MD3; Clahsen-van Groningen, Marian MD, PhD4; Haas, Mark MD, PhD5; Henriksen, Kammi J. MD6; Horsfield, Catherine MD3; Loupy, Alexandre MD7; Mengel, Michael MD8; Perkowska-Ptasińska, Agnieszka MD9; Rabant, Marion MD, PhD10; Racusen, Lorraine C. MD11; Solez, Kim MD8; Becker, Jan U. MD12 A 2018 Reference Guide to the Banff Classification of Renal Allograft Pathology, Transplantation: November 2018 – Volume 102 – Issue 11 – p 1795-1814 doi: 10.1097/TP.0000000000002366
C4d is the foot print of DSAs related AMR. It’s deposited and bound covalently to capillary endothelial basement membrane ,where DSAs -endothelial Antigen HLA and nonHLA ) interact.
Diffuse distribution is strongly associated with Capillaritis in the form of glomerulitis and peritubular capillaritis (g+ptc>2) referred to as Micro vascular inflimmation MVI,as per Banff classification.
Diffuse C4d staining is associated with high circulating DSAs titer .
It’s usually predisposed to, by preformed DSAs, those DSAs are Complement fixing and precipitate active AMR.
In the contrary AMR clinical phenotype dose not correlate with severity of underlying MVI.
Smoldering AMR:
In other cases C4d might be deposited focally , in association with mild MVI.
Focal C4d is detected in the following circumstances:
1) in asymptomatic patients ,on protocol biopsy
2) Normal allograft function.
3) No proteinuria.
4)Subclinical AMR.
4) It might be diffuse, but most commonly focal.
Etiology :
1) low circulating level of DSAs
2) Mostly non-complement fixing DSAs.
Significance and prognosis:
This focal C4d staining portend high risk for development of TG. As low grade MVI continued to trigger endothelial damage predisposing to TG and PTC multilayring.
TG is linked to C4d positivity.
It’s difficult and quite challenging to treate.
Negative C4d AMR:
In some cases of active and chronic active AMR, C4d staining is negative.
DSAs are positive.
Mechanism is Antibody mediated neutrophils and Natural killer cytotoxicity.
C4d is the foot print of DSAs related AMR. It’s deposited and bound covalently to capillary endothelial basement membrane ,where DSAs -endothelial Antigen HLA and nonHLA ) interact.
Diffuse distribution is strongly associated with Capillaritis in the form of glomerulitis and peritubular capillaritis (g+ptc>2) referred to as Micro vascular inflimmation MVI,as per Banff classification.
Diffuse C4d staining is associated with high circulating DSAs titer .
It’s usually predisposed to, by preformed DSAs, those DSAs are Complement fixing and precipitate active AMR.
In the contrary AMR clinical phenotype dose not correlate with severity of underlying MVI.
Smoldering AMR:
In other cases C4d might be deposited focally , in association with mild MVI.
Focal C4d is detected in the following circumstances:
1) in asymptomatic patients ,on protocol biopsy
2) Normal allograft function.
3) No proteinuria.
4)Subclinical AMR.
4) It might be diffuse, but most commonly focal.
Etiology :
1) low circulating level of DSAs
2) Mostly non-complement fixing DSAs.
Significance and prognosis:
This focal C4d staining portend high risk for development of TG. As low grade MVI continued to trigger endothelial damage predisposing to TG and PTC multilayring.
TG is linked to C4d positivity.
It’s difficult and quite challenging to treate.
Negative C4d AMR:
In some cases of active and chronic active AMR, C4d staining is negative.
DSAs are positive.
Mechanism is Antibody mediated neutrophils and Natural killer cytotoxicity.
C4d is the foot print of DSAs related AMR. It’s deposited and bound covalently to capillary endothelial basement membrane ,where DSAs -endothelial Antigen HLA and nonHLA ) interact.
Diffuse distribution is strongly associated with Capillaritis in the form of glomerulitis and peritubular capillaritis (g+ptc>2) referred to as Micro vascular inflimmation MVI,as per Banff classification.
Diffuse C4d staining is associated with high circulating DSAs titer .
It’s usually predisposed to, by preformed DSAs, those DSAs are Complement fixing and precipitate active AMR.
In the contrary AMR clinical phenotype dose not correlate with severity of underlying MVI.
Smoldering AMR:
In other cases C4d might be deposited focally , in association with mild MVI.
Focal C4d is detected in the following circumstances:
1) in asymptomatic patients ,on protocol biopsy
2) Normal allograft function.
3) No proteinuria.
4)Subclinical AMR.
4) It might be diffuse, but most commonly focal.
Etiology :
1) low circulating level of DSAs
2) Mostly non-complement fixing DSAs.
Significance and prognosis:
This focal C4d staining portend high risk for development of TG. As low grade MVI continued to trigger endothelial damage predisposing to TG and PTC multilayring.
TG is linked to C4d positivity.
It’s difficult and quite challenging to treate.
Negative C4d AMR:
In some cases of active and chronic active AMR, C4d staining is negative.
DSAs are positive.
Mechanism is Antibody mediated neutrophils and Natural killer cytotoxicity.
C4d is the foot print of DSAs related AMR. It’s deposited and bound covalently to capillary endothelial basement membrane ,where DSAs -endothelial Antigen HLA and nonHLA ) interact.
Diffuse distribution is strongly associated with Capillaritis in the form of glomerulitis and peritubular capillaritis (g+ptc>2) referred to as Micro vascular inflimmation MVI,as per Banff classification.
Diffuse C4d staining is associated with high circulating DSAs titer .
It’s usually predisposed to, by preformed DSAs, those DSAs are Complement fixing and precipitate active AMR.
In the contrary AMR clinical phenotype dose not correlate with severity of underlying MVI.
Smoldering AMR:
In other cases C4d might be deposited focally , in association with mild MVI.
Focal C4d is detected in the following circumstances:
1) in asymptomatic patients ,on protocol biopsy
2) Normal allograft function.
3) No proteinuria.
4)Subclinical AMR.
4) It might be diffuse, but most commonly focal.
Etiology :
1) low circulating level of DSAs
2) Mostly non-complement fixing DSAs.
Significance and prognosis:
This focal C4d staining portend high risk for development of TG. As low grade MVI continued to trigger endothelial damage predisposing to TG and PTC multilayring.
TG is linked to C4d positivity.
It’s difficult and quite challenging to treate.
Negative C4d AMR:
In some cases of active and chronic active AMR, C4d staining is negative.
DSAs are positive.
Mechanism is Antibody mediated neutrophils and Natural killer cytotoxicity.
Criteria for C4d staining is linear & circumferential deposition in at least 25% of cortex & medulla( excluding necrotic tissues). The distribution of the deposition can be classified into:
Diffuse defined as positive for C4d deposition, focal & minimal staining remain uncertain issue. But most expert consider focal staining red flag especially if DSA was positive.
C4d staining in PTC is highly specific for presence of DSA, so when deposition is diffuse mean there is strong correlation with anti-HLA Abs with poor graft outcome.
References:
Good
According to Banff guidelines significant C4d deposition in PCT is a prerequisite for diagnosis of ABMR
C4d deposition patters
1- diffuse pattern if involved more than 50 % of PTC , commonly associated with DSA presence and increased risk for ABMR and poor graft survival
2- focal C4d deposition if involve (10-50%) of PTC : usally no increased risk of ABMR
3-Minimal: if less than deposition involve less than 10 %
4- c4d negative ABMR : no C4d deposition but associated with histological evidence of tissue damage and presence of DSA as in case of non complement fixing Ab mediated rejection or aoolantibody against FC receptors on NK cells
Worthington JE, McEwen A, McWilliam LJ, Picton ML, Martin S. Association between C4d staining in renal transplant biopsies, production of donor-specific HLA antibodies, and graft outcome. Transplantation. 2007;83(4):398-403. doi:10.1097/01.tp.0000251430.11723.b6
☆ C4d Stain Interpretation:
C4d0—No staining of PTC and medullary vasa recta (0%).
C4d1—Minimal C4d staining (>0 but 50% of PTC and medullary vasa recta).
C4d2—Focal C4d staining (10-50% of PTC and medullary vasa recta).
C4d3—Diffuse C4d staining (>50% of PTC and medullary vasa recta).
☆ Banff Lesion Score C4d :
The score evaluates the extent of staining for C4d on endothelial cells of PTCs and medullary vasa recta by IF on snap frozen sections of fresh tissue or IHC on formalin-fixated and paraffin-embedded tissue.
Scoring of C4d staining is based on the percentage of peritubular capillaries and vasa recta that has a linear, circumferential staining pattern.
The minimal sample for evaluation is 5 high-power fields of cortex and/or medulla without scarring or infarction. C4d must not be scored in areas of infarction.
In terms of extent of staining, with IF, Banff Lesion Score C4d ≥ 2 is considered positive and a criterion for antibody interaction with tissue and as equivalent to DSA, whereas with IHC, Banff Lesion Score C4d ≥ 1 is counted as positive already.
Diffuse C4d staining pattern is significantly associated with the presence of circulating donor-specific antibodies and higher risk of ABMR.
—————-
Reference
(1) Cornell LD (2021) Histopathologic Features of Antibody Mediated Rejection: The Banff Classification and Beyond. Front. Immunol. 12:718122. doi: 10.3389/fimmu.2021.718122
(2) Roufosse, C., Simmonds, N., Clahsen-van Groningen, M., Haas, M., Henriksen, K. J., Horsfield, C., Loupy, A., Mengel, M., Perkowska-Ptasińska, A., Rabant, M., Racusen, L. C., Solez, K., & Becker, J. U. (2018). A 2018 Reference Guide to the Banff Classification of Renal Allograft Pathology. Transplantation, 102(11), 1795–1814. https://doi.org/10.1097/TP.0000000000002366
Complement component 4 fragment d (C4d) is a degradation product of complement component C4, which can bind covalently to the endothelial cell surface.
Positive immunostaining indicates local complement activation and is scored with Banff Lesion Score C4d.
The clinical significance of these findings is different in grafts exposed to anti-blood group antibodies (ABO-incompatible allografts), in which isoagglutinin-mediated complement activation does not appear to be injurious to the graft and may represent accommodation.
However, when caused by donor-specific antibodies against HLA or other antigens, it may indicate AMR activity. Based on the high specificity of C4d positivity, it is considered equivalent to the serological proof of donor-specific antibody (DSA) for a diagnosis of AMR, although it is still recommended to test for DSA.
Banff Lesion Score C4d.
A, IHC staining
An example of C4d3, this image demonstrates linear and circumferential staining of endothelial cells in virtually all peritubular capillaries.
B, IF staining for C4d.
This image shows an example of a Banff Lesion Score of C4d3; using IF, a minimum score of C4d ≥ 2 is considered positive.
C4d scoring –
Demonstration of PTC C4d deposition by immunohistochemistry was considered positive if grade was >C4d0.
Score of C4d1, C4d2, and C4d3 was given if 1–9% (minimal staining), 10%–50% (focal), and >50% PTCs were involved (diffuse), respectively.
C4dnegative ABMR was defined as biopsies with C4d <2 on immunofluorescence or C4d >0 on IHC, ptc> 0 and g >0, g+ptc ≥2, or g>0 or ptc >0 and acute TMA in the absence of any other cause of TMA(1).
C4d staining predict presence of DSA with sensitivity more than 95%..
In general C4d positive has a poor graft survival than C4d negative(2)
Detection of intragraft complement activation has strong independent value as an additional indicator of ABMR associated with adverse kidney transplant outcomes(2).
The worst 8-year death-censored graft survival was observed in patients scored as C4d3 ,followed by C4d2 , C4d1 , and C4d0 respectively (2)(figure2).
References:
1-BANFF classification 2017.
2- Željko Kikić, Alexander Kainz et al, Capillary C4d and Kidney Allograft Outcome in Relation to Morphologic Lesions Suggestive of Antibody-Mediated Rejection, CJASN August 2015, 10 (8) 1435-1443; DOI: https://doi.org/10.2215/CJN.09901014.
C4D refer to complement split product of the classic complement pathway activation by antibodies antigen allo-immune response on the endothelial surfaces its essential marker for the diagnosis of the AMR since 1990s and IHC staining for C4Ddeposition in PTCs is one’s diagnostic criteria for AMR, Ca ABMR according to Banff histological diagnostic criteria scores since 2003, 2005 and update in 2017. with high specificity but restricted sensitivity in the era of C4D negative complement independent ABMR .
C4D staining:
-Glomerular deposition (mesangial and vascular pole) of C4D can be seen in normal kidney
-Diffuse staining defines by ≥ 50% of linear deposition of C4D in PTCS which is specific for the diagnosis of AMR.
-Focal staining defined by < 50% of C4D +ve in PTCS, when the biopsy performed in early stage of ABMR as the DSA complement activation proceed the C4D split product disposition , so still can be corelated with ABMR .
-Little or negative C4D staining in PTCS with typical histological evidence of ABMR and positive DSA can be seen in CD4 negative complement Independent ABMR, with or without DSA, usually late onset, more with Class 11 denovo DSA or non HLA antibodies , progressive but less aggressive course and poor response to anti-rejection therapy as its association with chronic transplant glomerulopathy and progressive graft loss, using molecular gene endothelial transcript ( ENDAT) which is molecular marker of endothelial cellular activation and injury can confirm the diagnosis
-C4D detection by either using immuno-histochemical staining (IHC) on formalin embedded paraffin sections with positive score >0, and ≥1% positive c4d staining of the PTC, its more specific but less sensitive compared to IF staining.
IF staining by using fresh frozen section with scoring of C4d is significant if 10% and above.
-C4d positive alone without evidence of endothelial injury is still of uncertain significance and it’s not enough to diagnosis AMR, like in case of ABOI transplant which indicate the graft accommodation.
-Molecular gene expression analysis in C4d+ve without evidence of allograft injury has the potential to identify patients at higher risk of developing ABMR (2).
Also the correlation of C4D staining with DSA positive assay for the diagnosis of ABMR can be challenged in case of low titer of DSA below the detection level or due to presence of non-HLA abs like ATIR antibodies , anti-endothelial ABs , which can be seen in aggressive type of ABMR with c4d +VE ,DSA -ve
References:
1- Haas M. The significance of C4d staining with minimal histologic abnormalities. Curr Opin Organ Transplant. 2010 Feb;15(1):21-7. doi: 10.1097/MOT.0b013e3283342ebd. PMID: 19907328.
2- Dominy KM, Willicombe M, Al Johani T, Beckwith H, Goodall D, Brookes P, Cook HT, Cairns T, McLean A, Roufosse C. Molecular Assessment of C4d-Positive Renal Transplant Biopsies Without Evidence of Rejection. Kidney Int Rep. 2018 Sep 18;4(1):148-158. doi: 10.1016/j.ekir.2018.09.005. PMID: 30596178; PMCID: PMC6308373.
3-kidney transplant in adult: clinical features and diagnosis of acute renal allograft rejection: up to date 2022.