Dear All Thank you for your replies. I enjoyed reading them. In addition to the sensitivity (IF) and specificity (IHC), Each technique has advantages and disadvantages. Regarding assessing the glomeruli for C4d staining, IF give positive control making it not suitable to assess the glomeruli. Both techniques are required to have the full picture especially if C4d staining was negative on IHC. IF is always one step ahead
Thanks prof explanation, Let us remember IF is done on frozen section while IHC requires formalin-fix parrafin-embedded tissue. IHC may be technically more difficult and the cost may go up
but for cost effectiveness can we start with with immunofluorescence staining ? if it is positive with high intensity ptcC4d mor than 10 percent it is ok, but if it is negative or equivocal so go ahead for IHC ?
Nazik Mahmoud
2 years ago
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?
Although the IF is very sensitive and specific but we need fresh tissue to get the result but IHC is highly specific and we can use paraffin block to get the results
But the IF is preferable than IHC
Dalia Eltahir
2 years ago
Peritubular capillary complement 4d staining is one of the criteria for the diagnosis of antibody-mediated rejection .Staining by two method : The immu-nofluorescence technique applied to frozen sections The immunohistochemistry technique applied to paraffin-embedded tissue.immunohistochemistry staining caused more dif-ficulties in interpretation, due to nonspecific staining in tubular cells and surrounding interstitium.Study by ROC curve showed immunohistochemistry has a speci-ficity of 100% and a sensitivity of 81.2% in relation to immunofluorescence .The immunohistochemistry method presents an excellent specificity but lower sensitivity to C4d detection in allograft dysfunction. The evalu-ation is more difficult, requiring a more experienced observer than the immunofluorescence method.
Wee Leng Gan
2 years ago
Yes. For highly sensitive and specific test result for c4d staining.
Reference
1)Nickeleit V, Mihatsch MJ. Kidney transplants, antibodies and rejection: is C4d a magic marker? Nephrol Dial Transplant. 2003;18:2232–2239.
Nandita Sugumar
2 years ago
IF AND IHC FOR C4D STAINING
IF is more sensitive
Cut off point for IHC for c4d positivity is not standardized or established
IHC with cut off of 10% can predict adverse graft outcome and can guide therapy
C4d staining is not sensitive marker for transplant glomerulopathy.
IF – frozen, IHC – paraffin base
IF uses monoclonal antibody and IHC uses polyclonal antibody
Both methods are needed to assess C4d staining accurately and predict graft outcome and graft injury.
REFERENCES
Crary GS, Raissian Y, Gaston RC, Gourishankar SM, Leduc RE, Mannon RB, Matas AJ, Grande JP. Optimal cutoff point for immunoperoxidase detection of C4d in the renal allograft: results from a multicenter study. Transplantation. 2010 Nov 27;90(10):1099-105. doi: 10.1097/TP.0b013e3181f7fec9. PMID: 21430605; PMCID: PMC3171966.
Ahmed Fouad Omar
2 years ago
Do we need both Immuno-peroxidase (IHC) and Immunofluorescence for C4d testing? Why?
·The answer depends on the pathology lab infra-structure and the pathologist experience. But whenever possible combining both techniques can yield both sensitive and specific results specially if the IHC result of C4d is negative
· In the renal allograft, C4d can be detected can be by both Immunofluorescence staining or Immunohistochemistry(immune peroxidase) technique.
· Each technique has its own pros and cons
· Immunofluorescence (IF):Still the gold standard, scored as C4d2 & C4d3 in Banff scoring system. Pros:
§ More sensitive
§ simple
§ Lower cost
§ Gives faster results.
Cons:
§ Less specific
§ False positive staining of the glomeruli.
§ Done on frozen sections, so, it requires 2 core, one for LM and another for immunofluorescence
§ Cannot be stored for later assessment
· IHC(Immune peroxidase):widely used, Score C4d >0 in Banff scoring system Pros:
§ More specific, no positive staining of the glomeruli.
§ Can be stored for later assessment of samples with good preservation of histological features
§ Can use the same sample required for LM(can use small biopsy tissue) Cons:
§ less sensitive
§ More time consuming
§ requires special storage, thus more costly
References:
Khairwa. A. The Relevance of Complement C4d Staining in Renal Allograft Biopsies. Indian Journal of Transplantation, 2020;14(2).
H Ludovico-Martins , C Silva et al, (Analysis of different staining techniques for c4d detection in renal allograft biopsies) Transplant Proc. 2009 Apr;41(3):862-5
Hamdy Hegazy
2 years ago
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why? It is advised to do both methods to achieve the advantages of both, However, higher cost will be a restricting factor.
Immune-fluorescence (IF) advantages are higher sensitivity, easier, quicker, cheaper and gives positive control in the glomerulus.
Immune-fluorescence (IF) disadvantages include: needs fresh frozen sample, less specific, false positive staining of glomeruli, requires fluorescent microscope and can’t evaluate the glomerulus.
Immunoperoxidase (IHC) advantages are higher specificity, ability to evaluate the glomerulus, samples can be preserved and can be done on paraffin sections. Immunoperoxidase disadvantages include: lower sensitivity, higher cost, more difficult technique and lacking of positive control.
If I were asked to choose only one of them, I would prefer IHC because it could be done on paraffin section and doesn’t require more tissues.
IF is still the gold standard
so it’s better to do both to get the advantages of both techniques but due to the cost barrier immunoperoxidase is preferred because it’s done over paraffin section and no extra tissue is needed
Akram Abdullah
2 years ago
4. Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?both can detect C4d , & both can give complete picture of what happened , if one was the only option , I will start with IHC if PTC positive for c4d , it is enough for the diagnosis of positive C4d .
Ramy Elshahat
2 years ago
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?c4d staining is considered as evidence of AB injury which is now one of the cornerstones in the diagnosis of ABMR and sometimes it’s your only evidence when DSA can’t be detected so it’s a very important test that usually affects your management.
there are 2 methods to detect it and each method has its own advantage and disadvantage immunofluorescence
adv.
very sensitive and presented early
easy
cheep
giving +ve control in the glomerulus
disadv.
done on a fresh frozen sample
glomerulus can’t be evaluated
less specific
immunoperoxidase
adv.
very specific
glomerulus can be evaluated
done on paraffin section so no more tissue is needed
disadv.
less sensitive
expensive
more difficult in technique
no +ve control
so it’s better to do both to get the advantages of both techniques but due to the cost barrier immunoperoxidase is preferred because it’s done over paraffin section and no extra tissue is needed
ahmed saleeh
2 years ago
Immunofluorescence technique:
Is a sensitive approach applied on fresh frozen sections , positive control in glomerular. C4d 2 or 3 (>10%) is considered significant .
Immunohistochemistry technique:
Is a less sensitive but has more specificity where fixed paraffin sections are used .C4d >0 is significant .
So to conclude IF is more sensitive but needs extra sample fresh frozen one
Abdullah Raoof
2 years ago
The central diagnostic criterion for humoral rejection/ABMR is the demonstration of C4d in peritubular capillaries (PTCs) and vasa recta. C4d deposition in PTC is the most specific indicator of the presence of circulating DSA and its interaction with endothelial cells in the graft. There is a strong correlation between the presence of C4d deposition and circulating anti-donor HLA antibodies
The sensitivity of IF using a monoclonal antibody to C4d in frozen sections is greater than immunoperoxidase stains using the polyclonal antibody in FFPE tissue.
Staining for C4d by IF was described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to the 2003 Banff conference. Medullary vessels are typically positive and can be the only place of C4d positivity in some cases with marked edema and cortical injury.
In IHC, strong staining is usually not seen as tissue pretreatment influences staining intensity. IHC demonstrated a substantially lower prevalence and extent of C4d deposition in PTC and had a lower reproducibility than IF.
In one study . On average, the estimated area of C4d-positive PTC in the diffuse group was 36% lower by IHC than by IF.
Another study demonstrated that IHC has acceptable sensitivity and specificity, as compared with IF (the overall specificity of the IHC method compared with IF was 98%, and sensitivity was 87.5%).
Sometimes, the plasma in the capillaries is fixed by the formalin processing and also stains for C4d by IHC, which interferes with interpretation.
Extravasation of C4d into the connective tissue is also common and should not be mistaken for capillary wall deposition. Hence, intraluminal and interstitial C4d may also be seen and is an artifact of fixation.
The most sensitive method for C4d is the 3-step indirect IF on frozen sections using one of the monoclonal antibodies. However, many prefer to use the 2-step indirect IF method with the monoclonal antibody because of
· its simplicity,
· quick turnaround time,
· and relatively low cost.
Although more sensitive, IF requires extra biopsy sample (other than FFPE tissue) and frozen sections facility .
As IHC is feasible in FFPE tissue, it can be easily done from biopsy tissue submitted for light microscopy study when frozen section facility (for IF) is not available or when extra biopsy tissue is not available for frozen sections.
C4d can be detected in mesangial regions by IF (not IHC) in patients with no rejection. In the absence of PTC C4d staining, these isolated glomerular deposits are nondiagnostic for ABMR.
According to above mentioned information, to answer the question ,
I think to start with IF ( as it is simple ,low cost, not time consuming )if it is negative this will excludes ABMR ,if it is positive (GRADE 4- diffuse linear ) this is diagnostic of ABMR . A situation in between these two needs further assessment with IHC .
REFRENCE
1- Etta PK. C4d staining and antibody-mediated rejection in renal transplantation: Current status. Indian J Transplant 2020;14:197-201.
2- Seemayer CA, Gaspert A, Nickeleit V, Mihatsch MJ. C4d staining of renal allograft biopsies: A comparative analysis of different staining techniques. Nephrol Dial Transplant 2007;22:568-76.
3- Troxell ML, Weintraub LA, Higgins JP, Kambham N. Comparison of C4d immunostaining methods in renal allograft biopsies. Clin J Am Soc Nephrol 2006;1:583-91.
4- Nadasdy GM, Bott C, Cowden D, Pelletier R, Ferguson R, Nadasdy T. Comparative study for the detection of peritubular capillary C4d deposition in human renal allografts using different methodologies. Hum Pathol 2005;36:1178-85.
AMAL Anan
2 years ago
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?-there are two techniques for c4d identification which indude :
*Immenofterorscence ( IF ) and immunohistochemistry ( IHC )
Both techniques have advantages and disadvantages:
A- immunofluroscence :
c4d staining in IF has the following features :-
* it applied to frozen sections widespread, strong linear circumferential peritubular capillaries staining in cortex or medulla ( exclude scar or necrotic area ).
* positive control in glomeruli.
* c4d can be detected in mesangial region inn patient with no rejection.
* it is more sensitive.
* c4d2 or 3 > 10% is significant and require feozen section facility
B- immunohistochemistry
c4d statin in immunohistochemistry had the following features:
it is applied to formalin fixed paraffin embedded tissue crisp diffuse liner continuous ingredient around in the PTC wall finely granular pattern on high power
Considering all the pros and cons it would be best to have a combination of both a technique but if only one to be chosen, IHC would be better due to no need of extra tissue ( formalin fixed sample can be used).
References: 1) 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. 2) Khairwa A. The relevance of complement C4d staining in renal allograft biopsies. Indian J Transplant 2020;14:94-8. 3) Etta PK. C4d staining and antibody-mediated rejection in renal transplantation: Current status. Indian J Transplant 2020;14:197-201.
Balaji Kirushnan
2 years ago
There are 3 methods of detection of C4d in renal allograft biopsy…
Immunofluorescence technique using 2 step technique…This is a simple, quick method of detection of C4d…The 2 step is an indirect IF technique a monoclonal antibody against the C4d is used and the staining is picked up….It is a very sensitive technique. The disadvantage is that it needs another extra biopsy tissue to be sampled and submitted in the michelle’s fixative and not in Formalin
Immunofluorescence technique using 3 step technique…This is done on frozen tissue and is the most sensitive of all…But this is time consuming than the 2 step IF technique…Staining for C4d is described as widespread strong linear circumferential PTC staining in cortex or medulla according to 2003 Banff criteria. Medullary PTC also are a place of common C4d staining.. This technique also required another extra core of the renal tissue to be submitted..
Immunohistochemistry using formalin fixed tissue by polyclonal antibodies – generally called the IHC technique…The sensitivity of the IHC by using polyclonal antibody is less sensitive than IF towards detection of C4d…The strong staining is not seen in IHC as pretreatment influences staining intensity…IHC demonstrated a lower reproducibility of C4d deposition in the PTC and was less sensitive…in one study, only 69% of diffuse and 13% of focal C4d expressing cases were in line classified by IF and IHC. On average, the estimated area of C4d-positive PTC in the diffuse group was 36% lower by IHC than by IF…there is another study which shows comparable sensitivity between IHC and IF in terms of C4d staining…..There would be a lot of background staining in the IHC treated group due to pre treatment with formailin or paraffin embeddment….the advantage is that even one tissue is would be suffice to process for the C4d staining by IHC….
MICHAEL Farag
3 years ago
Yes!
Sensitivity of immunoflorescence using monoclonal antibody to C4d in frozen section is greater than immunoperoxidase stain using polyclonal antibody in formalin fixed paraffin embedded tissue.
Immunohistochemistry is specific method than immunoflorescence using polyclonal antibody.
In addition to the sensitivity (IF) and specificity (IHC), Each technique has advantages and disadvantages. Regarding assessing the glomeruli for C4d staining, IF give positive control making it not suitable to assess the glomeruli.
Both techniques are required to have the full picture especially if C4d staining was negative on IHC.
Ahmed Omran
3 years ago
IF:
Tissue : frozen section requiring extra tissues for examination and frozen section facility.
Staining :C4d staining is wide spread, strong linear circumferential PTC staining in cortex & medulla.
Scoring :scored as C4d2 & C4d3;significant scoring 10% or more.
Sensitivity: high sensitivity.
IHC:
Tissue :used with paraffin-embedded tissue.
Staining : described as crisp, linear continuous diffuse and lying around PTC wall.
Scoring :Score C4d >0.
Specificity: highly specific, useful in small biopsy tissue, using tissue examined by LM.
_good preservation of histological features ( important in scoring non atrophic tissue) which is difficult in IF.
_ longer time than IF and need extra control; more cost.
To increase sensitivity and specificity of C4d staining, it is preferred to use both techniques. References:
Khairwa. A. The Relevance of Complement C4d Staining in Renal Allograft Biopsies. Indian Journal of Transplantation, 2020;14(2).
Troxell M., Wientraub L., Higgins J. and Kambham N. Comparison of C4d Immunostaining Methods in Renal Allograft Biopsies.Clin J Am Nephrol,2006;1:583-591.
Drtalib Salman
3 years ago
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why? YES for negative patient but for positive patient ex: with IHC (highly specific) Noneed to do immunofluorescence .
ptc C4d consider finger print of Antibody mediated rejection so we need to carefully look for it in renal biopsy
2 method to diagnose :
Immunoperoxidase (IHC):
it is use small piece of tissue biopsy not exclude dead necrotic tissue ,highly specific more than 95 percent but disadvantage expensive ,time consuming low sensitivity 85 percent .
Immunofluorescence:
highly sensitive ,low specific , exclude dead tissue, need large piece frozen section .detect glomerular staining .
Theepa Mariamutu
3 years ago
Do we need both immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?
Yes both,for better interpretation
IHC done by polyclonal antiserum on paraffin embedded tissue sections with IHC detections
IF is monoclonal C4d antibody on frozen sections with IF detections
Both method has comparable results
Nadasdy et al reported both methods are comparable too
IHC has 98% specificity and 87.5% sensitivity
Advantages of IHC
Performed on serial levels of the same tissue that was cut for LM
Offered generous tissue sample for analysis with good preservation of histologic features
Exclusion of the strophic areas of cortex is difficult with IF
Disadvantages of IHC
Background staining must taken into account
Small percentage of of cases might be false negative by either method
references
Megan L. Troxell, Lauren A. Weintraub, John P. Higgins and Neeraja Kambham. Comparison of C4d Immunostaining Methods in Renal Allograft Biopsies
Anju Khairwa* The Relevance of Complement C4d Staining in Renal Allograft Biopsies
Department of Pathology, ESIC Model Hospital, Gurugram, Haryana, India
Immunofluorescence (IF)
1- Applied for frozen section
2- C4d staining is described as widespread, strong linear circumferential PTC staining excluding area of scar or necrosis
3- Requires extra tissue (large sample)
4- More sensitive
5- Need frozen section facility
6- Scoring is significant if C4d2 or C4d3
7- Less costly
Immunohistochemistry (IHC)
1- Paraffin-embedded tissue
2- C4d is crisp, linear, continuous, diffuse, and lying around in PTC wall. Its strength is slighter and changeable. Have a non-specific fine granular pattern
3- Can be applied to small sample
4- More specific
5- Needs extra control
6- Scoring is significant if C4d > 0
7- Costly
8- time-consuming
CARLOS TADEU LEONIDIO
3 years ago
Yes, its necessary. Its because C4d detection by IHC is more specific and C4d detection by IF is more sensitive.
Reference:
– Khairwa A. The relevance of complement C4d staining in renal allograft biopsies. Indian J Transplant 2020;14:94-8.
MOHAMED Elnafadi
3 years ago
Immunostaining of renal allograft biopsies for C4d deposition has become an important diagnostic tool in the recognition of humoral-mediated graft rejection.
Differences in staining properties are multifactorial, including tissue preparation (e.g., transport media, fresh frozen versus formalin-fixed, paraffin-embedded),
The sensitivity and the specificity of the IHC method is acceptable in comparison with the IF method. Although at the present time IF is considered the “gold standard,”to detect c4d staining.the follow-up graft function in many cases does not show any trend to suggest superiority of positive IF or IHC staining.IHC offered a generous tissue sample for analysis, with good preservation of histologic features, which is important for scoring nonatrophic areas in biopsy. Exclusion of atrophic areas of cortex in scoring IF samples shows more difficulty to demonstrate.
Shereen Yousef
3 years ago
There are diffrent methods used for C4d detection which use either frozen tissue for immunofluorescence (IF) (monoclonal antibodies are more commonly used than polyclonal antibodies) or formalin-fixed paraffin-embedded (FFPE) tissue for immunohistochemistry (IHC) (using polyclonal antibodies).
The sensitivity of IF using a monoclonal antibody to C4d in frozen sections is greater than immunoperoxidase stains using the polyclonal antibody in FFPE tissue.
Another study demonstrated that IHC with anti-C4d polyclonal antibody has acceptable sensitivity and specificity, as compared with IF staining with the monoclonal antibody (the overall specificity of the IHC method compared with IF was 98%, and sensitivity was 87.5%).
Sometimes, the plasma in the capillaries is fixed by the formalin processing and also stains for C4d by IHC, which interferes with interpretation.
Extravasation of C4d into the connective tissue is also common and should not be mistaken for capillary wall deposition. Hence, intraluminal and interstitial C4d may also be seen and is an artifact of fixation.
Nonspecific background staining is also common in IHC. The most sensitive method for C4d is the 3-step indirect IF on frozen sections using one of the monoclonal antibodies. However, many prefer to use the 2-step indirect IF method with the monoclonal antibody because of its simplicity, quick turnaround time, and relatively low cost.
so every method still have advantages and disadvantages and using both if possible will improve accuracy of diagnosis.
Praveen Kumar Etta.C4d staining and antibody-mediated rejection in renal transplantation: Current status.COMMENTARY,Year : 2020 | Volume : 14 | Issue : 3 | Page : 197-201
Abdulrahman Ishag
3 years ago
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?
There are three methods used for C4d detection which use either frozen tissue for immunofluorescence (IF) ( using monoclonal antibodies ) or formalin-fixed paraffin-embedded (FFPE) tissue for immunohistochemistry (IHC) (using polyclonal antibodies).
The sensitivity of IF using a monoclonal antibody to C4d in frozen sections is greater than immunoperoxidase stains using the polyclonal antibody in FFPE tissue.
The use of indirect IF method with the monoclonal antibody is preferred because of its simplicity, quick turnaround time, and relatively low cost. Although more sensitive, IF requires extra biopsy sample (other than FFPE tissue) and frozen sections facility.
As IHC is feasible in FFPE tissue, it can be easily done from biopsy tissue submitted for light microscopy study when frozen section facility (for IF) is not available or when extra biopsy tissue is not available for frozen sections.
Reference ;
1-Nadasdy GM, Bott C, Cowden D, Pelletier R, Ferguson R, Nadasdy T. Comparative study for the detection of peritubular capillary C4d deposition in human renal allografts using different methodologies. Hum Pathol 2005;36:1178-85.
2-Haas M, Loupy A, Lefaucheur C, Roufosse C, Glotz D, Seron D, et al. The Banff 2017 kidney meeting report: Revised diagnostic criteria for chronic active T cell-mediated rejection, antibody-mediated rejection, and prospects for integrative endpoints for next-generation clinical trials. Am J Transplant 2018;18:293-307.
Huda Al-Taee
3 years ago
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?
Yes, we need it as every method has its own advantages and limitations and the biopsy sample does not always have enough tissues for frozen sections.
Advantages of IF:
high sensitivity
exclude necrotic tissues
lower intraobserver variability
Disadvantages of IF:
need frozen sections
need large volume tissues.
glomerular staining as a control
less specific
Advantages of IHC:
excellent specificity.
feasible in paraffin-embedded, formalin-fixed tissue.
Disadvantages of IHC:
lower sensibility.
non-specific staining.
cost and time preparation.
need for external controls.
References;
Khairwa A. The Relevance of Complement C4d Staining in Renal Allograft Biopsies. Indian Journal of Transplantation. 2020; 14:94-8.
Sousa H. C4d detection in renal allograft biopsies: immunohistochemistry vs. immunofluorescence. Port J Nephrol Hypert 2012; 26(4): 272-277.
Seemayer ChA, Gaspert A, Nickeleit V, Mihatsch MJ. C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques. Nephrol Dial Transplant (2007) 22: 568–576.
Troxell ML, Weintraub LA, Higgins JP, Kambham N. Comparison of C4d Immunostaining Methods in Renal Allograft Biopsies. Clin J Am Soc Nephrol. 2006; 1: 583–591.
Mahmud Islam
3 years ago
Both IHC and IF are useful
IF entails the usage of fresh samples, here we can make use of IHC to evaluate paraffin fixed samples available even retrospectively
for diagnosis of AMR we need tubulitis, c4d staining in PTC not glomerules; IF stain glomerulus as well
IF is more specific but we need to evaluate in the same day of staining opposite to IHC which can be evaluated many times without loss of staining pattern
Positive C4d immunostaining in peritubular capillaries of renal transplant biopsies has been incorporated into the criteria for the diagnosis of humoral rejection. Study by Megan and colleagues confirm that normal glomeruli show mesangial C4d staining with an IF detection method applied to frozen tissue but not with the polyclonal antibody applied to formalin-fixed, paraffin-embedded tissue (IHC method).
The sensitivity and the specificity of the IHC method is acceptable in comparison with the IF method. Although at the present time IF is considered the “gold standard,”
IHC offers several advantages. IHC is performed on serial levels of the same tissue that is cut for light microscopy; therefore, IHC offered a generous tissue sample for analysis, with good preservation of histologic features, which is important for scoring nonatrophic areas.
I think IHC alone can serve the purpose
We don’t need both IHC and IF for c4d.
IF for c4d requires-
1.extra specimen
2.frozen section
But it is more sensitive than IHC with cheap and easy to perform and time saving.
Whereas IHC requires-
1.no extra specimen
2.performed in formalin fixed paraffin embeddment with greater clarity in ultrastructural component,but less sensitive,time consuming,more costlier than IF study.
So if IF will miss c4d, IHC will not help anyway and if IF will find c4d then IHC may find c4d,so to diagnose abmr any way we require tissue injury+c4d+dsa.So when we are suspecting rejection IF is far better than IHC .
Only in rebiopsy of graft and only one tissue availability, IHC will definitely help.
I think NO
as immunoflorecent has more senstivity than IHC but it need fresh spacemen
IHC using stain in formalin fixed paraffin embedded tissue.so no need for fresh spacemen but less senstivity so if IF done no need for IHC
Sahar elkharraz
3 years ago
Yes!
Sensitivity of immunoflorescence using monoclonal antibody to C4d in frozen section is greater than immunoperoxidase stain using polyclonal antibody in formalin fixed paraffin embedded tissue.
Immunohistochemistry is specific method than immunoflorescence using polyclonal antibody.
References
Praveen Kumar Etta et al. C4d staining and antibody-mediated rejection in renal transplantation: Current status:Department of Nephrology and Renal Transplantation, Virinchi Hospitals and Max Superspeciality Medical Centre, Hyderabad, Telangana, Indan; Year : 2020 | Volume : 14 | Issue : 3 | Page : 197-201.
*Immunostaining of renal allograft biopsies for C4d deposition has become an important diagnostic tool in the recognition of humoral-mediated graft rejection.
*IF C4d staining is more sensitive than IHC
specificity of the IHC method compared with IF was 98%, and sensitivity was 87.5%
The sensitivity and the specificity of the IHC method is acceptable in comparison with the IF method. Although at the present time IF is considered the “gold standard
*IHC offers several advantages.
IHC generally had higher background staining but better morphologic preservation
IHC was performed on serial levels of the same tissue that was cut for light microscopy so
IHC offered a generous tissue sample for analysis, with good preservation of histologic features, which is important for scoring nonatrophic areas.
IHC staining of levels cut in parallel with tissue for light microscopy permits saving of labor and tissue and results in a permanent archival slide record.
*The disadvantage of C4d staining by IHC are lower sensibility, nonspecific background staining, more costly and more time consuming, and it needs external controls.
*In IF exclusion of atrophic areas of cortex samples was significantly more difficult.
Morphologic analysis of frozen tissue sections is not optimal, and strong tubular basement membrane labeling occasionally hampered evaluation of peritubular capillary staining with the IF method. Although it was possible, with experience, to distinguish areas of atrophy by faint tubular basement membrane IF staining, this proved considerably more difficult than in the IHC stained samples.
*a small percentage of cases may be falsely negative by either staining method; therefore, Careful correlation of C4d results, donor-specific serum testing, and histologic features should be performed in each case and further correlated with clinical history.
1-Megan L. Troxell, Lauren A. Weintraub, John P. Higgins and Neeraja Kambham. Comparison of C4d Immunostaining Methods in Renal Allograft Biopsies
2-Anju Khairwa* The Relevance of Complement C4d Staining in Renal Allograft Biopsies
Department of Pathology, ESIC Model Hospital, Gurugram, Haryana, India
YES we do , The sensitivity of IF using a monoclonal antibody to C4d in frozen sections is greater than immunoperoxidase stains using the polyclonal antibody in FFPE tissue. Staining for C4d by IF was described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to the 2003 Banff conference. In IHC, strong staining is usually not seen as tissue pretreatment influences staining intensity. IHC demonstrated a substantially lower prevalence and extent of C4d deposition in PTC and had a lower reproducibility than IF. In one study, only 69% of diffuse and 13% of focal C4d expressing cases were in line classified by IF and IHC. On average, the estimated area of C4d-positive PTC in the diffuse group was 36% lower by IHC than by IF. Another study demonstrated that IHC with anti-C4d polyclonal antibody has acceptable sensitivity and specificity, as compared with IF staining with the monoclonal antibody (the overall specificity of the IHC method compared with IF was 98%, and sensitivity was 87.5%).
Etta PK. Comprehensive management of the renal Transplant recipient. Indian J Transplant 2019;13:240-51. Seemayer CA, Gaspert A, Nickeleit V, Mihatsch MJ. C4d staining of renal allograft biopsies: A comparative analysis of different staining techniques. Nephrol Dial Transplant 2007;22:568-76.
-Peritubular capillary complement 4d staining is one of the criteria for the diagnosis of antibody-mediated rejection, and research into this is essential to kidney allograft evaluation. The immunofluorescence technique (IF) was applied to frozen sections while the immunohistochemistry technique(IHC) was applied to paraffin-embedded tissue. Several studies showed :
– the three-step IF method appeared to be the most sensitive and IHC the less sensitive.
-The IF method as the gold standard, found that the IHC specificity was 98% and sensitivity 87.5%. There is difficulty in interpretation of IHC, which is due to unspecific background staining.
-On average, the degree of C4d staining with an IHC method was lower by about a degree, which means that many diffusely staining cases in IF method turned focally positive in the IHC method.
– In the presence of glomerular damage strong C4d staining of the glomerular basement membrane was detected by both methods. The endothelia of arteries and arterioles were sometimes positive.
-Studies recommended the use of the IF method in frozen tissue. IHC advantages
1- Excellent specificity.
2 – Feasible in formalin-fixed, paraffin-embedded tissue. FFPE is the usual tissue processing for kidney biopsies, so this material is always available. The IHC technique could be used when no frozen tissue is accessible. IHC disadvantages
1. Lower sensibility. Although IHC presents an excellent specificity, it has a lower sensitivity than IF. Some false negatives were registered. The positive staining area by IHC is in some cases lower than by IF cases. Results obtained with IHC must be interpreted with some reserve since clinical relevance has only been established for IF results, and focal staining in IHC can correspond to diffuse staining in IF, for example.
2. Nonspecific staining. nephropathologists stated that IHC staining causes more difficulties in interpretation, due to the existence of unspecific staining in tubular cells and surrounding interstitium.
3 . Cost and time preparation. The IHC technique was more expensive than IF. The IHC method takes over one hour more to complete than the IF technique. The IF technique applied to frozen sections does not need previous processing and can be finished in one hour. By the IF method, the results can be available to medical staff less than two hours after tissue reception in the laboratory.
4 .Need for external controls.
–The immunofluorescence technique applied to frozen sections is the gold-standard method for complement C4d staining and the immunohistochemistry technique can safely be used when immunofluorescence is not available. Reference: –Ana Santos1, Helena Viana1, Maria João Galvão1, et al.C4d detection in renal allograft biopsies: immunohistochemistry vs. immunofluorescence.Port J Nephrol Hypert 2012; 26(4): 272-277
For the above mentioned advantages and disadvantages both tests are needed and that will increase both sensitivity and specificity. We can overcome the disadvantages of both tests by doing both of them.
Dear All We need to review together the sensitivity and specificity of both techniques. Please write to us which one is more sensitive and which one is more specific.
IF study is more sensitive (specificity is 93% and sensitivity 94%).In the contrary,for IHC its more specific, as any positive result more than 0% considered as positive with specificity of 98% and sensitivity of 87%) formalin fixed, paraffin embeded specimen reduced sensitivity in IHC in comparison to frozen section in IF .IF would give false positive stainig in glomeruli.
Reference:
1)Lecture of prof .Halawa
2)Comparison of C4d immunostaining methods Renal allograft biopsies. Megan L et al.CJASN 2006
Glomerular mesangial staining was shown in frozen sections while in paraffin-embedded tissues it was along the glomerular capillary wall.
Amit Sharma
3 years ago
Two techniques utilized for C4d identification include immunofluorescence (IF) and immunohistochemistry (IHC). Both the techniques have their own advantages and limitations.
A) Immunoflourescence (IF): C4d staining in IF has following features – (1,2)
It is applied to frozen sections
Widespread, strong linear circumferential PTC staining in cortex or medulla (excluding scar/necrotic area).
Positive control in glomeruli
C4d can be detected in mesangial region in patients with no rejection (3)
It is more sensitive.
C4d2 or 3 (>10%) is significant
It requires extra tissue
It requires frozen section facility
B) Immunohistochemistry (IHC): C4d staining in IHC has following features – (1,2)
It is applied to formalin fixed, paraffin embedded tissue.
Crisp, diffuse, linear continuous, lying around in the PTC wall; finely granular pattern on high power.
More specific
C4d>0 (>1%) is significant
Useful when tissue is not available for frozen section or the biopsy tissue is small.
It has lower sensitivity
It has non-specific background staining
No positive control in glomeruli.
It needs external control
It has higher cost
It is more time consuming
A study compareing IF and IHC concluded that IF was better suited for diagnostic and research purposes as C4d detection by IHC was lower. (4)
Probably, this is the reason that in C4d>0 is significant in IHC whereas in IF, C4d 2or 3 is deemed significant.
Considering all the pros and cons, it would be best to have a combination of both the techniques. But If only one needs to be chosen, IHC would be better due to no need of extra tissue (formalin fixed sample can be used).
References:
1) 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. 2) Khairwa A. The relevance of complement C4d staining in renal allograft biopsies. Indian J Transplant 2020;14:94-8. 3) Etta PK. C4d staining and antibody-mediated rejection in renal transplantation: Current status. Indian J Transplant 2020;14:197-201.
4) Seemayer CA, Gaspert A, Nickeleit V, Mihatsch MJ. C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques. Nephrol Dial Transplant. 2007 Feb;22(2):568-76. doi: 10.1093/ndt/gfl594. Epub 2006 Dec 12. PMID: 17164320.
Thank you Sir.
Immunofluorescence is more sensitive.
Doaa Elwasly
3 years ago
No it is not needed
There are advantages to both methods for detection of C4d – for example, it is recognized that detection of C4d by immunofluorescence provides the most sensitive and specific marker for C4d positivity . However, immunofluorescence studies require additional tissue, which may not be practical for protocol biopsies or for retrospective studies. Meanwhile, there is considerable variation between centers on thresholds for “focal” or “diffuse” positive staining, both by immunofluorescence and by immunoperoxidase, rendering it difficult to compare results for C4d positivity between centers. (1)
Ludovico-Martins H .et al demonstrated that frozen-IHC and paraffin-IF can be considered alternative techniques to frozen-IF for C4d detection. The paraffin-IHC technique displayed the lowest concordance rate for C4d detection.(2)
The sensitivity of IF using a monoclonal antibody to C4d in frozen sections is greater than immunoperoxidase stains using the polyclonal antibody in formalin-fixed paraffin-embedded FFPE tissue. Staining for C4d by IF was described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to the 2003 Banff conference. Medullary vessels are typically positive and can be the only place of C4d positivity in cases with marked edema and cortical injury. In IHC, strong staining is usually not seen as tissue pretreatment influences staining intensity. IHC demonstrated a substantially lower prevalence and extent of C4d deposition in PTC and had a lower reproducibility than IF.
The most sensitive method for C4d is the 3-step indirect IF on frozen sections using one of the monoclonal antibodies. However, many prefer to use the 2-step indirect IF method with the monoclonal antibody because of its simplicity, shorter time, and relatively low cost.
Although more sensitive, IF requires extra biopsy sample (other than FFPE tissue) and frozen sections facility. As IHC is feasible in FFPE tissue, it can be easily done from biopsy tissue submitted for light microscopy study when frozen section facility (for IF) is not available or when extra biopsy tissue is not available for frozen sections. (3)
Reference
1- Crary G Set al .Optimal Cut-off Point for Immunoperoxidase Detection of C4d in the Renal Allograft: Results from a Multicenter Study .Transplantation. 2010 ; 90(10): 1099–1105.
2-Ludovico-Martins H .et al .Analysis of Different Staining Techniques for C4d Detection in Renal Allograft Biopsies.Transplantation Proceedings 2009 ;41(3):862-865. 3-Kumar Etta P.C4d staining and antibody-mediated rejection in renal transplantation: Current status.Indian Journal of Transplanation 2021;14(3):197-201.
Well done Doaa Based on what you have written, we need both of them. Do not forget, IF give positive control in the glomeruli
Tahani Ashmaig
3 years ago
☆Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing?
▪︎ My answer is No
Notes:
________
C4d staining:
▪︎Currently, there are 3 commercially available antibodies for stainingof C4d. Two of these antibodies are monoclonal and are usually used with either a 3- or a 2-step indirect immunofluorescence (IF) methodology on frozen sections [1].
and a polyclonal antibody used on formalin-fixed, paraffin-embedded tissue section with an immunoperoxidase detection system [1].
☆So to answer the question Why?
▪︎All methodologies and antibodies tested provided adequate results with only minor differences between them[2], so we don’t need need both IHC and IF for C4d testing
▪︎The sensitivity of IF using a monoclonal antibody to C4d in frozen sections is greater than immunoperoxidase stains ( the most sensitive is the 3. steps IF).
▪︎The 2-step indirect IF method with the 2. steps is preferable because of its simplicity, quick turnaround time, and relatively low cost.
▪︎
▪︎In IHC, strong staining is usually not seen as tissue pretreatment influences staining intensity.
▪︎IHC demonstrated a substantially lower prevalence and extent of C4d deposition in PTC and had a lower reproducibility than IF [2].
▪︎Sometimes, the plasma in the capillaries is fixed by the formalin processing and also stains for C4d by IHC, which interferes with interpretation[2]
▪︎Nonspecific background staining is common in IHC.
▪︎ As IHC is feasible in FFPE tissue, it can be easily done from biopsy tissue submitted for light microscopy study when frozen section facility (for IF) is not available or when extra biopsy tissue is not available for frozen sections.
▪︎C4d can be detected in mesangial regions by IF (not IHC) in patients with no rejection.
______________________________
Ref
[1] Gyongyi M Nadasdy et al. “Comparative study for the detection of peritubular capillary C4d deposition in human renal allografts using different methodologies”. Hum Pathol. 2005 Nov.
[2] Praveen K.et al “C4d staining and antibody-mediated rejection in renal transplantation: Current status” Volume : 14 | Issue : 3 | Page : 197-201
The answer is No, we don’t need to do both IHC and IFC(Troxell et al., 2006).
Immunofluorescence is still considered the gold standard.
The sensitivity and specificity of Immunoperoxidase is 87.5% and 97% respectively.
IHC has acceptable sensitivity and specificity as compared to immunofluorescence.
IF is performed on frozen sections of tissue that were transported in saline, stained with monoclonal ant-C4d antibody. IHC performed on paraffin-embedded tissue stained with polyclonal C4d antiserum.
TROXELL, M. L., WEINTRAUB, L. A., HIGGINS, J. P. & KAMBHAM, N. 2006. Comparison of C4d Immunostaining Methods in Renal Allograft Biopsies. Clinical Journal of the American Society of Nephrology, 1, 583-591.
1-IHC: It is possible to store pieces of paraffin-embedded tissue for further evaluation (IHC offered a good tissue sample for analysis, with good preservation of histologic features )
The method is more specific than IF, although it is less sensitive (there is no positive staining of Glomeruli) according to Banff’s score, with c4d > 0 it is statistically significant.
The process is more time-intensive (during the preparation phase) and there are issues in interpreting
Positive findings for C4d in paraffin do not rule out the possibility of positive results in frozen material.
2- IF is still the gold standard because it is based on frozen sections.
Allows for the quick and sensitive detection of C4d positive at a lower cost and with greater sensitivity.
Disadvantages
Specificity has been reduced ( False positive staining of glomeruli on the background )
C4d was found in the mesangium of the glomerulus in frozen native specimens, but not in paraffin sections that had been exposed to light for a brief period of time (and therefore could not be preserved for subsequent evaluation).
Ref: H Ludovico-Martins, C Silva et al, (Analysis of different staining techniques for c4d detection in renal allograft biopsies)
Immunostaining of renal allograft biopsy for C4d deposition has become an important diagnostic tool in the recognition of humoral – mediated graft rejection.
Presence of C4d deposition in PTC have correlated with poor graft outcome.
Detection of C4d deposition in PTC have incorporated into Banff 97 classification system as criteria for diagnosis of humoral rejection in renal allograft.
C4d is a fragment of the classical complement pathway,C4 is activated by Ag-Ab complex.,C4 is activated and cleaved into C4a ans c4b then c4b cleavage into c4c and c4d,this fragment contain the covalent bond to the tissue and remains at the site of complement activation for longer period in contrast with the complement fragment.
tow technigues have been used to detected c4d immunostaining in the allofraft biopsy IMMUNOFLUORESCENCE
It is gold standard method
more sensitive than IHC
staining intensity affect by length of time c4d was present at Ag/Ab reaction site
applied to frozen section of fresh tissue
positive result indicated by linear staining within preitublar capillaries of cortex and or medulla,glomerular staining is not specific , IMMUNOHISTOCHEMISTRY
technique applied to paraffin -embedded tissue of renal allograft biopsy.
disadvantage :
difficult in interpretation due to nonspecific staining in tubular cells and interstitium
more specificity but lower sensitivity to c4d detection in allograft dysfunction.
regarding if we need both technique,no .as we mention above the IF is first option if not available ,IHC can be used
why?because some of studies shows that all cases negative by IF also negative by IHC and all positive cases by IF are positive by IHC
Reference:
1) Immunoadsorption in severe C4d-positive acute kidney allograft rejection: a randomized controlled trial.
Böhmig GA, Wahrmann M, Regele H, Exner M, Robl B, Derfler K, Soliman T, Bauer P, Müllner M, Druml W
Am J Transplant. 2007;7(1):117. Epub 2006 Nov 15.
2) Feucht HE: Complement C4d in graft capillaries: The missing link in recognition of humoral alloreactivity. Am J Transplant3 :646– 652,2003 3) Abbas AK, Lichtman AH, Pober JS: Transplantation immunology. In: Cellular and Molecular Immunology, 4th ed., edited by Abbas AK, Lichtman AH, Pober JS, Philadelphia, W.B. Saunders Co.,2000 , pp369– 385
done on frozen section so need extra tissues for examination & frozen section facility.
C4d staining described as wide spread, strong linear circumferential PTC staining in cortex & medulla.
scored as C4d2 & C4d3.
significant scoring 10% or more.
high sensitivity.
IHC:
used with paraffin-embedded tissue.
staining described as crisp, linear continuous diffuse & lying around PTC wall.
Score C4d >0.
highly specific.
useful in small biopsy tissue, use same tissue examined by LM.
good preservation of histological features ( important in scoring non atrophic tissue) which is difficult in IF.
cost, need longer time than IF & need extra control.
So to increase sensitivity & specificity of C4d staining it is better to use both techniques.
References:
Khairwa. A. The Relevance of Complement C4d Staining in Renal Allograft Biopsies. Indian Journal of Transplantation, 2020;14(2).
Troxell M., Wientraub L., Higgins J. and Kambham N. Comparison of C4d Immunostaining Methods in Renal Allograft Biopsies.Clin J Am Nephrol,2006;1:583-591.
In order to detect C4d in renal allograft biopsy’s two techniques have been developed these include immunfluorescence technique and immunohistochemistry , Each technique has its own pros and cons.
Immunofluorescence technique
This technique is more rapid and more sensitive and gold standard . It basically involves measurement of standing intensity which is calculated by the length of time C4d was present at antigen and antibody binding location however it is a disadvantage that samples can deteriorate quickly. This cannot be used for assessment at a later time.
Immunohistochemistry
This techniques is more specific but costly and time consuming. It is used when frozen sections facility is not available or the sample tissue is very small rendering it unsuitable for frozen section. Feasible in formalin fixed paraffin embedded tissues.
Both techniques can be used for glomerular staining however IF is more sensitive and associated with positive controls .
Referrence-
Ana Santos et al. C4d detection in renal allograft biopsies. immunohistochemistry vs immune fluorescence. Port.J Nephrol hypert.2012;24/6: 272-277.
Renal biopsy is gold standard for diagnosis of
Acute rejection in renal transplant.
C4d Staining is a useful adjacent marker of the humoral limb of rejection boht in early
and late post transplant period.
Method : using two methods
immunohistochemistry and 1.immunofluorescencetechnique:
A.Feasble in formalin-fixed paraffin-embedded tissue.
B.The immunohistochemistry metho presents an excellent specificity but lower sensitivity to C4d detection in allograft dysfunction.
C.The evaluation is more difficult, requiring a more experienced observer .
D. Difficulties in interpretation, due to nonspecific stainingin tubular cells and surrounding interstitium.
E. cost and time preparations
F.Need for external control.
G.IHC offers several advantages. it was performed on serial levels of the same tissue therefore, it offers a generous tissue sample for analysis, with good preservation of histologic features, which is important for scoring nonatrophic areas. Exclusion of atrophic areas of cortex in scoring IF samples was significantly more difficult. Automated IHC staining of levels cut in parallel with tissue for light microscopy permits saving of labor and tissue and results in a permanent archival slide record.
.Based on these , we conclude that the immu-nohistochemistry technique can safely be used when immunofluorescence is not available
2. immunoflourecence (IF) staining need adequate fresh tissues in frozen section is more sensitive for C4D detection but can give false postive staining in glomeruli , its rapid test and should be interpreted at same time , less cost but less specific compared to IHC .
Ref:
1.C4d Detection in Renal Allograft Biopsies: Immunohistochemistry vs. ImmunofluorescenceA. Santos, H. Viana, +2 authors F. Nolasco Published 1 October 2012Biology, Medicine portuguese journal of nephrology and hypertension.
2.
Comparison of C4d Immunostaining Methods in Renal Allograft Biopsies
Megan L. Troxell, Lauren A. Weintraub, John P. Higgins and Neeraja Kambham
CJASN May 2006, 1 (3) 583-591; DOI: https://doi.org/10.2215/CJN.00900805
Yes, both needed for C4D testing in ABMR and its part of daignositic banff criteria .The IHC staining usually done in formalin fixed and paraffin-embedded tissue even small biopsy or inadequate tissue can be used but need external control, more time and more cost , IHC testing very specific for C4D staining in ABMR .HC staining need experities in interpretaion due to the nonspecific staining background in the tubules and interstitium.
immunoflourecence (IF) staining need adequate fresh tissues in frozen section is more sensitive for C4D detection but can give false postive staining in glomeruli , its rapid test and should be interpreted at same time , less cost but less specific compared to IHC .
IHC can be safely used with 100% specifcity if the IF staining not available(2).
References:
1-The relevance of complement C4d staining in renal allograft biopsies
Anju Khairwa:Indian Journal of Transplantation, Year 2020, Volume 14, Issue 2 [p. 94-:98.
2-C4d Detection in Renal Allograft Biopsies: Immunohistochemistry vs. ImmunofluorescenceA. Santos, H. Viana, +2 authors F. Nolasco Published 1 October 2012Biology, Medicine portuguese journal of nephrology and hypertension.
Two techniques are mostly used for identification of C4d IHC and IF in allograft biopsies of the kidney .
IF technique applied to frozen sections whereas IHC technique used to paraffin embedded tissue used routinely in various laboratories .
C4d is significant staining in 1% or more of the PTC for formalin/IHC IP, or 10% or more for frozen/frequency IF.
Immunofluorescence
More sensitive method.
Measure the staining intensity calculated by the length of time C4d was present at the Ag/Ab reaction site
Quicker than Immunochemistry.
Rapid deterioration of the samples.
advantages of IHC
specific no false positive
avialble formalin-fixed, paraffin-embedded tissue
disadvantages of IHC:
high cost than IF
need processing and takes much time than IF
less sensitive than IF e.g focal c4d stain by IHC can be diffuse c4d stain by IF, so we can get false negative result by IHC
Need for external control.
The IHC method for C4d detection feasible in formalin fixed, paraffin embedded tissue, it is used when frozen sections facility not available, IHC also useful when small biopsy tissue or tissue not available for frozen sections .
Last edited 3 years ago by MOHAMMED GAFAR medi913911@gmail.com
Two techniques are mostly used for identification of C4d in allograft biopsies of the kidney:
1. Indirect immunofluorescence (IF):
applied to frozen sections
In acute and chronic AMR‐positive C4d stain with IF technique is described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to a consensus at 2003 Banff Conference.
The scoring of C4d is significant by IF on frozen section ie C4d2 or C4d3
C4d is significant staining in 10% or more for frozen/frequency
C4d detection by IF is more sensitive.
Less expensive .
Rapid detection of C4d positivity.
IF method for C4d requires extra tissue and frozen sections facility.
Can not be stored for latter assessment.
Still the gold standard technique
2. Immunohistochemical (IHC):
IHC technique used to paraffin‐embedded tissue used routinely in various laboratories.
C4d staining is crisp, linear, continuous, diffuse, and lying around in the PTC wall, while the strength typically is slighter and changeable, but it may have a finely granular pattern in high power.
The scoring of C4d is significant by IHC C4d >0 on paraffin sections.
C4d detection by IHC more specific
The IHC method for C4d detection feasible in formalin‐fixed, paraffin‐embedded tissue, it is used when frozen sections facility not available, IHC also useful when small biopsy tissue or tissue not available for frozen sections.
The disadvantage of C4d staining by IHC are lower sensibility, nonspecific background staining, more costly and more time consuming, and it needs external controls.
IHC method Widely available.
Uses the same sample that is processed for LM .
Reference:
The Relevance of Complement C4d Staining in Renal Allograft Biopsies
Anju Khairwa*
Department of Pathology, ESIC Model Hospital, Gurugram, Haryana, India.
o IF (more sensitive), but mostly requires fresh tissue in frozen section, excess tissue and must be visualized immediately after staining.
o IHC (immune-histochemistry or immune-peroxidase), can be done on paraffin sections. Can be interpreted at any time but less sensitive than IF.
o The disadvantage of IHC are lower sensitivity, nonspecific background staining, more expensive and time consuming, and it needs external controls
o C4d deposits may be linear, circumferential or crisps. It is dynamic and can vary.
o C4d in IF appearance is “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” (2003 Banff ), while in IHC, C4d staining is crisp, linear, continuous, diffuse, and lying around in the PTC wall (less strength but has granular pattern in high power).
· According Banff diagnostic criteria:
o C4d staining in PTCs by IHC on paraffin sections (at least 10% PTC positive is considered as significant) as it is less sensitive.
o In contrast, by IFs on frozen sections Banff scores C4d2 or C4d3 is considered significant (more sensitive).
I think, better to use the IF for its sensitivity and low cost, as long as available fresh tissue for frozen section. If not available, we can do IHC. However, also IF can be done nowadays on paraffin section by special technique from available paraffin blocks.
in conclusion, each technique has its pros and cons. However, we can depend on IF alone to detect c4d without need to IHC, but not the reverse.
C4d deposition can be measured by
1- Immunefluresnt (IF) using monoclonal or polyclonal Ab on frozen tissue
· Advantages of IF
more sensitive than IHC ( both 2 and 3 step indirect IF on frozen section using monoclonal Ab)
and 2 step method is easier , takes shorter time and relatively lower cost
· Disadvatages
Requires extra biopsy tissue and frozen section facility
2- Immunehistochemistery (IHC) examination of formalin fixed paraffin embded tissue
Advantages of IHC
· doesn’t require extra biopsy tissue as can be done on biopsy sample prepared for LM
Disadvantages
· Less sensitive than IF
· Tissue pretreatment affects intensity of C4d staining
3 step indirect IF on frozen section is the gold standard test for detecting C4d deposits but if un available IHC has comperable sensitivity
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?
Methods used for C4d detection which use :
Frozen tissue for immunofluorescence (IF) (monoclonal antibodies are more commonly used than polyclonal antibodies.) Formalin-fixed paraffin-embedded (FFPE) tissue for
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?
Ithink we need to do both
# Study condected that : normal glomeruli show mesangial C4d staining with an IF detection method applied to frozen tissue but not with the polyclonal antibody applied to formalin fixed, paraffin embedded tissue (IHC method)
#The sensitivity and the specificity of the IHC method is acceptable in comparison with the IF method.
# Although at the present time IF is considered the gold standard IF/ IHC discordant cases were seen in both no treatment and aggressive therapy groups the follow up graft function in these cases does not show any trend to suggest superiority of positive IF or IHC staining.
# In renal biopsy practice, IHC offers several advantages it was performed on serial levels of the same tissue that was cut for light microscopy, therefore IHC offered a generous tissue sample for analysis with good preservation of histologic features which is important for scoring nonatrophic areas.
# Exclusion of atrophic areas of cortex in scoring IF samples was significantly more difficult.
# So careful correlation of C4d results, donor-specific serum testing, and histologic features should be performed in each case and further correlated with clinical history.
References
Abbas AK, Lichtman AH, Pober JS: Transplantation immunology. In: Cellular and Molecular Immunology, 4th ed., edited by Abbas AK, Lichtman AH, Pober JS, Philadelphia, W.B. Saunders Co.,2000 , pp369– 385Google Scholar
Mauiyyedi S, Colvin RB: Humoral rejection in kidney transplantation: New concepts in diagnosis and treatment. Curr Opin Nephrol Hypertens11 :609– 618,2002
CrossRefPubMedGoogle Scholar
Bohmig GA, Exner M, Watschinger B, et.al Regele H: Acute humoral renal allograft rejection. Curr Opin Urol12 :95– 99,2002
4. Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?
Each one has its own pros & cons 1. Indirect immunofluorescence (IF):
– Uses monoclonal anti-C4d antibodies on frozen tissue.
– Relatively simple, highly sensitive, & specific
– Disadvantages include: i. Requires a frozen section ii. Requires a sample different from the one used for LM, thus less satisfactory correlation. iii. Requires fluorescent microscope. 2. Immunohistochemical (IHC):
– Uses formalin-fixed paraffin-embedded sections with polyclonal anti-C4d antibodies.
– Widely available.
– Uses the same sample that is processed for LM so no need for a second core.
– Several studies showed high sensitivity & specificity
– Disadvantage: leads to a loss of sensitivity of C4d of about 30%, compared to IF on frozen tissue. References 1. Bowdler AL, Griffiths DF, Newman GR. The morphological and immunohistochemical analysis of renal biopsies by light and electron microscopy using a single processing method. Histochem J 1989;21:393-402. 2. Troxell ML, Weintraub LA, Higgins JP, Kambham N. Comparison of C4d immunostaining methods in renal allograft biopsies. Clin J Am Soc Nephrol 2006;1:583-91. file:///C:/Users/TOSHIBA/AppData/Local/Temp/msohtmlclip1/01/clip_image001.gif 3. Santos A, Viana H, Galvão MJ, Carvalho F, Nolasco F. C4d detection in renal allograft biopsies: Immunohistochemistry vs. immunofluorescence. Port J Nephrol Hypertens 2012;26:272-7. 4. Mauiyyedi S, Crespo M, Collins AB et al. Acute humoral rejection in kidney transplantation: II. Morphology, immunopathology, and pathologic classification. J Am Soc Nephrol 2002; 13:779-787. 5. Bohmig GA, Exner M, Habicht A et al. Capillary C4d deposition in kidney allografts: a specific marker of alloantibody-dependent graft injury. J Am Soc Nephrol 2002; 13:1091-1099.
Both techniques are very useful in the detection C4d with different sensitivity and specificity
First IHC :
based on Paraffin-embedded sections
can be saved for later assessment (IHC offered a good tissue sample for analysis, with good preservation of histologic features )
More specific than IF however less sensitivity (No positive staining of Glomeruli )
according to Banff’s score with c4d > 0 it’s significant However disadvantages: less sensitive than IF
more Expensive
More time consuming ( during Preparation ) and difficulties in interpretation
Negative findings for C4d in paraffin do not exclude positive findings in frozen material.
2- IF Still gold Standard based on frozen sections
Allows rapid and sensitive detection of C4d positivity
less expensive
more sensitive
Disadvantages
Less specific ( False positive staining of glomeruli on the background )
C4d was expressed in the mesangium of the glomerulus in frozen native specimen but not in paraffin sections
of short time ( can’t be stored for later assessment )
Both techniques can be used (to improve of both sensitivity and specificity )
using double staining of immunofluorescence (C4d/CD34 double-immunofluorescence ) improves the detection of C4d with increasing specificity
Ref : C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques Christian A. SeemayerAriana Gaspert, Volker Nickeleit, Michael J. Mihatsch
, C4d/CD34 double-immunofluorescence staining of renal allograft biopsies for assessing peritubular capillary C4d positivity
Kuang-Yu Jen1 , Thuy B Nguyen1 , Flavio G Vincenti 2 and Zoltan G Laszik
Dear All
Thank you for your replies. I enjoyed reading them. In addition to the sensitivity (IF) and specificity (IHC), Each technique has advantages and disadvantages. Regarding assessing the glomeruli for C4d staining, IF give positive control making it not suitable to assess the glomeruli.
Both techniques are required to have the full picture especially if C4d staining was negative on IHC. IF is always one step ahead
Thanks prof explanation,
Let us remember IF is done on frozen section while IHC requires formalin-fix parrafin-embedded tissue.
IHC may be technically more difficult and the cost may go up
but for cost effectiveness can we start with with immunofluorescence staining ? if it is positive with high intensity ptcC4d mor than 10 percent it is ok, but if it is negative or equivocal so go ahead for IHC ?
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?
Although the IF is very sensitive and specific but we need fresh tissue to get the result but IHC is highly specific and we can use paraffin block to get the results
But the IF is preferable than IHC
Peritubular capillary complement 4d staining is one of the criteria for the diagnosis of antibody-mediated rejection .Staining by two method : The immu-nofluorescence technique applied to frozen sections The immunohistochemistry technique applied to paraffin-embedded tissue.immunohistochemistry staining caused more dif-ficulties in interpretation, due to nonspecific staining in tubular cells and surrounding interstitium.Study by ROC curve showed immunohistochemistry has a speci-ficity of 100% and a sensitivity of 81.2% in relation to immunofluorescence .The immunohistochemistry method presents an excellent specificity but lower sensitivity to C4d detection in allograft dysfunction. The evalu-ation is more difficult, requiring a more experienced observer than the immunofluorescence method.
Yes. For highly sensitive and specific test result for c4d staining.
Reference
1)Nickeleit V, Mihatsch MJ. Kidney transplants, antibodies and rejection: is C4d a magic marker? Nephrol Dial Transplant. 2003;18:2232–2239.
IF AND IHC FOR C4D STAINING
REFERENCES
Do we need both Immuno-peroxidase (IHC) and Immunofluorescence for C4d testing? Why?
·The answer depends on the pathology lab infra-structure and the pathologist experience. But whenever possible combining both techniques can yield both sensitive and specific results specially if the IHC result of C4d is negative
· In the renal allograft, C4d can be detected can be by both Immunofluorescence staining or Immunohistochemistry(immune peroxidase) technique.
· Each technique has its own pros and cons
· Immunofluorescence (IF): Still the gold standard, scored as C4d2 & C4d3 in Banff scoring system.
Pros:
§ More sensitive
§ simple
§ Lower cost
§ Gives faster results.
Cons:
§ Less specific
§ False positive staining of the glomeruli.
§ Done on frozen sections, so, it requires 2 core, one for LM and another for immunofluorescence
§ Cannot be stored for later assessment
· IHC(Immune peroxidase): widely used, Score C4d >0 in Banff scoring system
Pros:
§ More specific, no positive staining of the glomeruli.
§ Can be stored for later assessment of samples with good preservation of histological features
§ Can use the same sample required for LM(can use small biopsy tissue)
Cons:
§ less sensitive
§ More time consuming
§ requires special storage, thus more costly
References:
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?
It is advised to do both methods to achieve the advantages of both, However, higher cost will be a restricting factor.
Immune-fluorescence (IF) advantages are higher sensitivity, easier, quicker, cheaper and gives positive control in the glomerulus.
Immune-fluorescence (IF) disadvantages include: needs fresh frozen sample, less specific, false positive staining of glomeruli, requires fluorescent microscope and can’t evaluate the glomerulus.
Immunoperoxidase (IHC) advantages are higher specificity, ability to evaluate the glomerulus, samples can be preserved and can be done on paraffin sections.
Immunoperoxidase disadvantages include: lower sensitivity, higher cost, more difficult technique and lacking of positive control.
If I were asked to choose only one of them, I would prefer IHC because it could be done on paraffin section and doesn’t require more tissues.
IF is still the gold standard
so it’s better to do both to get the advantages of both techniques but due to the cost barrier immunoperoxidase is preferred because it’s done over paraffin section and no extra tissue is needed
4. Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?both can detect C4d , & both can give complete picture of what happened , if one was the only option , I will start with IHC if PTC positive for c4d , it is enough for the diagnosis of positive C4d .
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?c4d staining is considered as evidence of AB injury which is now one of the cornerstones in the diagnosis of ABMR and sometimes it’s your only evidence when DSA can’t be detected so it’s a very important test that usually affects your management.
there are 2 methods to detect it and each method has its own advantage and disadvantage
immunofluorescence
adv.
disadv.
immunoperoxidase
adv.
disadv.
so it’s better to do both to get the advantages of both techniques but due to the cost barrier immunoperoxidase is preferred because it’s done over paraffin section and no extra tissue is needed
Immunofluorescence technique:
Is a sensitive approach applied on fresh frozen sections , positive control in glomerular. C4d 2 or 3 (>10%) is considered significant .
Immunohistochemistry technique:
Is a less sensitive but has more specificity where fixed paraffin sections are used .C4d >0 is significant .
So to conclude IF is more sensitive but needs extra sample fresh frozen one
The central diagnostic criterion for humoral rejection/ABMR is the demonstration of C4d in peritubular capillaries (PTCs) and vasa recta. C4d deposition in PTC is the most specific indicator of the presence of circulating DSA and its interaction with endothelial cells in the graft. There is a strong correlation between the presence of C4d deposition and circulating anti-donor HLA antibodies
The sensitivity of IF using a monoclonal antibody to C4d in frozen sections is greater than immunoperoxidase stains using the polyclonal antibody in FFPE tissue.
Staining for C4d by IF was described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to the 2003 Banff conference. Medullary vessels are typically positive and can be the only place of C4d positivity in some cases with marked edema and cortical injury.
In IHC, strong staining is usually not seen as tissue pretreatment influences staining intensity. IHC demonstrated a substantially lower prevalence and extent of C4d deposition in PTC and had a lower reproducibility than IF.
In one study . On average, the estimated area of C4d-positive PTC in the diffuse group was 36% lower by IHC than by IF.
Another study demonstrated that IHC has acceptable sensitivity and specificity, as compared with IF (the overall specificity of the IHC method compared with IF was 98%, and sensitivity was 87.5%).
Sometimes, the plasma in the capillaries is fixed by the formalin processing and also stains for C4d by IHC, which interferes with interpretation.
Extravasation of C4d into the connective tissue is also common and should not be mistaken for capillary wall deposition. Hence, intraluminal and interstitial C4d may also be seen and is an artifact of fixation.
The most sensitive method for C4d is the 3-step indirect IF on frozen sections using one of the monoclonal antibodies. However, many prefer to use the 2-step indirect IF method with the monoclonal antibody because of
· its simplicity,
· quick turnaround time,
· and relatively low cost.
Although more sensitive, IF requires extra biopsy sample (other than FFPE tissue) and frozen sections facility .
As IHC is feasible in FFPE tissue, it can be easily done from biopsy tissue submitted for light microscopy study when frozen section facility (for IF) is not available or when extra biopsy tissue is not available for frozen sections.
C4d can be detected in mesangial regions by IF (not IHC) in patients with no rejection. In the absence of PTC C4d staining, these isolated glomerular deposits are nondiagnostic for ABMR.
According to above mentioned information, to answer the question ,
I think to start with IF ( as it is simple ,low cost, not time consuming )if it is negative this will excludes ABMR ,if it is positive (GRADE 4- diffuse linear ) this is diagnostic of ABMR . A situation in between these two needs further assessment with IHC .
REFRENCE
1- Etta PK. C4d staining and antibody-mediated rejection in renal transplantation: Current status. Indian J Transplant 2020;14:197-201.
2- Seemayer CA, Gaspert A, Nickeleit V, Mihatsch MJ. C4d staining of renal allograft biopsies: A comparative analysis of different staining techniques. Nephrol Dial Transplant 2007;22:568-76.
3- Troxell ML, Weintraub LA, Higgins JP, Kambham N. Comparison of C4d immunostaining methods in renal allograft biopsies. Clin J Am Soc Nephrol 2006;1:583-91.
4- Nadasdy GM, Bott C, Cowden D, Pelletier R, Ferguson R, Nadasdy T. Comparative study for the detection of peritubular capillary C4d deposition in human renal allografts using different methodologies. Hum Pathol 2005;36:1178-85.
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?-there are two techniques for c4d identification which indude :
*Immenofterorscence ( IF ) and immunohistochemistry ( IHC )
Both techniques have advantages and disadvantages:
A- immunofluroscence :
c4d staining in IF has the following features :-
* it applied to frozen sections widespread, strong linear circumferential peritubular capillaries staining in cortex or medulla ( exclude scar or necrotic area ).
* positive control in glomeruli.
* c4d can be detected in mesangial region inn patient with no rejection.
* it is more sensitive.
* c4d2 or 3 > 10% is significant and require feozen section facility
B- immunohistochemistry
c4d statin in immunohistochemistry had the following features:
Considering all the pros and cons it would be best to have a combination of both a technique but if only one to be chosen, IHC would be better due to no need of extra tissue ( formalin fixed sample can be used).
References:
1) 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.
2) Khairwa A. The relevance of complement C4d staining in renal allograft biopsies. Indian J Transplant 2020;14:94-8.
3) Etta PK. C4d staining and antibody-mediated rejection in renal transplantation: Current status. Indian J Transplant 2020;14:197-201.
There are 3 methods of detection of C4d in renal allograft biopsy…
Yes!
Sensitivity of immunoflorescence using monoclonal antibody to C4d in frozen section is greater than immunoperoxidase stain using polyclonal antibody in formalin fixed paraffin embedded tissue.
Immunohistochemistry is specific method than immunoflorescence using polyclonal antibody.
In addition to the sensitivity (IF) and specificity (IHC), Each technique has advantages and disadvantages. Regarding assessing the glomeruli for C4d staining, IF give positive control making it not suitable to assess the glomeruli.
Both techniques are required to have the full picture especially if C4d staining was negative on IHC.
IF:
Tissue : frozen section requiring extra tissues for examination and frozen section facility.
Staining :C4d staining is wide spread, strong linear circumferential PTC staining in cortex & medulla.
Scoring :scored as C4d2 & C4d3;significant scoring 10% or more.
Sensitivity: high sensitivity.
IHC:
Tissue :used with paraffin-embedded tissue.
Staining : described as crisp, linear continuous diffuse and lying around PTC wall.
Scoring :Score C4d >0.
Specificity: highly specific, useful in small biopsy tissue, using tissue examined by LM.
_good preservation of histological features ( important in scoring non atrophic tissue) which is difficult in IF.
_ longer time than IF and need extra control; more cost.
To increase sensitivity and specificity of C4d staining, it is preferred to use both techniques.
References:
Khairwa. A. The Relevance of Complement C4d Staining in Renal Allograft Biopsies. Indian Journal of Transplantation, 2020;14(2).
Troxell M., Wientraub L., Higgins J. and Kambham N. Comparison of C4d Immunostaining Methods in Renal Allograft Biopsies.Clin J Am Nephrol,2006;1:583-591.
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?
YES for negative patient but for positive patient ex: with IHC (highly specific) No need to do immunofluorescence .
ptc C4d consider finger print of Antibody mediated rejection so we need to carefully look for it in renal biopsy
2 method to diagnose :
Immunoperoxidase (IHC):
it is use small piece of tissue biopsy not exclude dead necrotic tissue ,highly specific more than 95 percent but disadvantage expensive ,time consuming low sensitivity 85 percent .
Immunofluorescence:
highly sensitive ,low specific , exclude dead tissue, need large piece frozen section .detect glomerular staining .
Do we need both immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?
Yes both,for better interpretation
IHC done by polyclonal antiserum on paraffin embedded tissue sections with IHC detections
IF is monoclonal C4d antibody on frozen sections with IF detections
Both method has comparable results
Nadasdy et al reported both methods are comparable too
IHC has 98% specificity and 87.5% sensitivity
Advantages of IHC
Performed on serial levels of the same tissue that was cut for LM
Offered generous tissue sample for analysis with good preservation of histologic features
Exclusion of the strophic areas of cortex is difficult with IF
Disadvantages of IHC
Background staining must taken into account
Small percentage of of cases might be false negative by either method
references
Megan L. Troxell, Lauren A. Weintraub, John P. Higgins and Neeraja Kambham. Comparison of C4d Immunostaining Methods in Renal Allograft Biopsies
CJASN May 2006, 1 (3) 583-591; DOI: https://doi.org/10.2215/CJN.00900805
Anju Khairwa* The Relevance of Complement C4d Staining in Renal Allograft Biopsies
Department of Pathology, ESIC Model Hospital, Gurugram, Haryana, India
http://www.ijtonline.in on Monday, February 7, 2022, IP: 10.232.74.26]
Immunofluorescence (IF)
1- Applied for frozen section
2- C4d staining is described as widespread, strong linear circumferential PTC staining excluding area of scar or necrosis
3- Requires extra tissue (large sample)
4- More sensitive
5- Need frozen section facility
6- Scoring is significant if C4d2 or C4d3
7- Less costly
Immunohistochemistry (IHC)
1- Paraffin-embedded tissue
2- C4d is crisp, linear, continuous, diffuse, and lying around in PTC wall. Its strength is slighter and changeable. Have a non-specific fine granular pattern
3- Can be applied to small sample
4- More specific
5- Needs extra control
6- Scoring is significant if C4d > 0
7- Costly
8- time-consuming
Yes, its necessary. Its because C4d detection by IHC is more specific and C4d detection by IF is more sensitive.
Reference:
– Khairwa A. The relevance of complement C4d staining in renal allograft biopsies. Indian J Transplant 2020;14:94-8.
Immunostaining of renal allograft biopsies for C4d deposition has become an important diagnostic tool in the recognition of humoral-mediated graft rejection.
Differences in staining properties are multifactorial, including tissue preparation (e.g., transport media, fresh frozen versus formalin-fixed, paraffin-embedded),
The sensitivity and the specificity of the IHC method is acceptable in comparison with the IF method. Although at the present time IF is considered the “gold standard,”to detect c4d staining.the follow-up graft function in many cases does not show any trend to suggest superiority of positive IF or IHC staining.IHC offered a generous tissue sample for analysis, with good preservation of histologic features, which is important for scoring nonatrophic areas in biopsy. Exclusion of atrophic areas of cortex in scoring IF samples shows more difficulty to demonstrate.
There are diffrent methods used for C4d detection which use either frozen tissue for immunofluorescence (IF) (monoclonal antibodies are more commonly used than polyclonal antibodies) or formalin-fixed paraffin-embedded (FFPE) tissue for immunohistochemistry (IHC) (using polyclonal antibodies).
The sensitivity of IF using a monoclonal antibody to C4d in frozen sections is greater than immunoperoxidase stains using the polyclonal antibody in FFPE tissue.
Another study demonstrated that IHC with anti-C4d polyclonal antibody has acceptable sensitivity and specificity, as compared with IF staining with the monoclonal antibody (the overall specificity of the IHC method compared with IF was 98%, and sensitivity was 87.5%).
Sometimes, the plasma in the capillaries is fixed by the formalin processing and also stains for C4d by IHC, which interferes with interpretation.
Extravasation of C4d into the connective tissue is also common and should not be mistaken for capillary wall deposition. Hence, intraluminal and interstitial C4d may also be seen and is an artifact of fixation.
Nonspecific background staining is also common in IHC. The most sensitive method for C4d is the 3-step indirect IF on frozen sections using one of the monoclonal antibodies. However, many prefer to use the 2-step indirect IF method with the monoclonal antibody because of its simplicity, quick turnaround time, and relatively low cost.
so every method still have advantages and disadvantages and using both if possible will improve accuracy of diagnosis.
Praveen Kumar Etta.C4d staining and antibody-mediated rejection in renal transplantation: Current status.COMMENTARY,Year : 2020 | Volume : 14 | Issue : 3 | Page : 197-201
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?
There are three methods used for C4d detection which use either frozen tissue for immunofluorescence (IF) ( using monoclonal antibodies ) or formalin-fixed paraffin-embedded (FFPE) tissue for immunohistochemistry (IHC) (using polyclonal antibodies).
The sensitivity of IF using a monoclonal antibody to C4d in frozen sections is greater than immunoperoxidase stains using the polyclonal antibody in FFPE tissue.
The use of indirect IF method with the monoclonal antibody is preferred because of its simplicity, quick turnaround time, and relatively low cost. Although more sensitive, IF requires extra biopsy sample (other than FFPE tissue) and frozen sections facility.
As IHC is feasible in FFPE tissue, it can be easily done from biopsy tissue submitted for light microscopy study when frozen section facility (for IF) is not available or when extra biopsy tissue is not available for frozen sections.
Reference ;
1-Nadasdy GM, Bott C, Cowden D, Pelletier R, Ferguson R, Nadasdy T. Comparative study for the detection of peritubular capillary C4d deposition in human renal allografts using different methodologies. Hum Pathol 2005;36:1178-85.
2-Haas M, Loupy A, Lefaucheur C, Roufosse C, Glotz D, Seron D, et al. The Banff 2017 kidney meeting report: Revised diagnostic criteria for chronic active T cell-mediated rejection, antibody-mediated rejection, and prospects for integrative endpoints for next-generation clinical trials. Am J Transplant 2018;18:293-307.
Yes, we need it as every method has its own advantages and limitations and the biopsy sample does not always have enough tissues for frozen sections.
Advantages of IF:
Disadvantages of IF:
Advantages of IHC:
Disadvantages of IHC:
References;
Both IHC and IF are useful
IF entails the usage of fresh samples, here we can make use of IHC to evaluate paraffin fixed samples available even retrospectively
for diagnosis of AMR we need tubulitis, c4d staining in PTC not glomerules; IF stain glomerulus as well
IF is more specific but we need to evaluate in the same day of staining opposite to IHC which can be evaluated many times without loss of staining pattern
Thank You
Positive C4d immunostaining in peritubular capillaries of renal transplant biopsies has been incorporated into the criteria for the diagnosis of humoral rejection. Study by Megan and colleagues confirm that normal glomeruli show mesangial C4d staining with an IF detection method applied to frozen tissue but not with the polyclonal antibody applied to formalin-fixed, paraffin-embedded tissue (IHC method).
The sensitivity and the specificity of the IHC method is acceptable in comparison with the IF method. Although at the present time IF is considered the “gold standard,”
IHC offers several advantages. IHC is performed on serial levels of the same tissue that is cut for light microscopy; therefore, IHC offered a generous tissue sample for analysis, with good preservation of histologic features, which is important for scoring nonatrophic areas.
I think IHC alone can serve the purpose
Thank You
We don’t need both IHC and IF for c4d.
IF for c4d requires-
1.extra specimen
2.frozen section
But it is more sensitive than IHC with cheap and easy to perform and time saving.
Whereas IHC requires-
1.no extra specimen
2.performed in formalin fixed paraffin embeddment with greater clarity in ultrastructural component,but less sensitive,time consuming,more costlier than IF study.
So if IF will miss c4d, IHC will not help anyway and if IF will find c4d then IHC may find c4d,so to diagnose abmr any way we require tissue injury+c4d+dsa.So when we are suspecting rejection IF is far better than IHC .
Only in rebiopsy of graft and only one tissue availability, IHC will definitely help.
Thank You
I think NO
as immunoflorecent has more senstivity than IHC but it need fresh spacemen
IHC using stain in formalin fixed paraffin embedded tissue.so no need for fresh spacemen but less senstivity
so if IF done no need for IHC
Yes!
Sensitivity of immunoflorescence using monoclonal antibody to C4d in frozen section is greater than immunoperoxidase stain using polyclonal antibody in formalin fixed paraffin embedded tissue.
Immunohistochemistry is specific method than immunoflorescence using polyclonal antibody.
References
Praveen Kumar Etta et al. C4d staining and antibody-mediated rejection in renal transplantation: Current status:Department of Nephrology and Renal Transplantation, Virinchi Hospitals and Max Superspeciality Medical Centre, Hyderabad, Telangana, Indan; Year : 2020 | Volume : 14 | Issue : 3 | Page : 197-201.
Thank You
Yes both method
*Immunostaining of renal allograft biopsies for C4d deposition has become an important diagnostic tool in the recognition of humoral-mediated graft rejection.
*IF C4d staining is more sensitive than IHC
specificity of the IHC method compared with IF was 98%, and sensitivity was 87.5%
The sensitivity and the specificity of the IHC method is acceptable in comparison with the IF method. Although at the present time IF is considered the “gold standard
*IHC offers several advantages.
IHC generally had higher background staining but better morphologic preservation
IHC was performed on serial levels of the same tissue that was cut for light microscopy so
IHC offered a generous tissue sample for analysis, with good preservation of histologic features, which is important for scoring nonatrophic areas.
IHC staining of levels cut in parallel with tissue for light microscopy permits saving of labor and tissue and results in a permanent archival slide record.
*The disadvantage of C4d staining by IHC are lower sensibility, nonspecific background staining, more costly and more time consuming, and it needs external controls.
*In IF exclusion of atrophic areas of cortex samples was significantly more difficult.
Morphologic analysis of frozen tissue sections is not optimal, and strong tubular basement membrane labeling occasionally hampered evaluation of peritubular capillary staining with the IF method. Although it was possible, with experience, to distinguish areas of atrophy by faint tubular basement membrane IF staining, this proved considerably more difficult than in the IHC stained samples.
*a small percentage of cases may be falsely negative by either staining method; therefore, Careful correlation of C4d results, donor-specific serum testing, and histologic features should be performed in each case and further correlated with clinical history.
1-Megan L. Troxell, Lauren A. Weintraub, John P. Higgins and Neeraja Kambham. Comparison of C4d Immunostaining Methods in Renal Allograft Biopsies
CJASN May 2006, 1 (3) 583-591; DOI: https://doi.org/10.2215/CJN.00900805
2-Anju Khairwa* The Relevance of Complement C4d Staining in Renal Allograft Biopsies
Department of Pathology, ESIC Model Hospital, Gurugram, Haryana, India
http://www.ijtonline.in on Monday, February 7, 2022, IP: 10.232.74.26]
This is excellent
YES we do ,
The sensitivity of IF using a monoclonal antibody to C4d in frozen sections is greater than immunoperoxidase stains using the polyclonal antibody in FFPE tissue.
Staining for C4d by IF was described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to the 2003 Banff conference.
In IHC, strong staining is usually not seen as tissue pretreatment influences staining intensity. IHC demonstrated a substantially lower prevalence and extent of C4d deposition in PTC and had a lower reproducibility than IF.
In one study, only 69% of diffuse and 13% of focal C4d expressing cases were in line classified by IF and IHC. On average, the estimated area of C4d-positive PTC in the diffuse group was 36% lower by IHC than by IF.
Another study demonstrated that IHC with anti-C4d polyclonal antibody has acceptable sensitivity and specificity, as compared with IF staining with the monoclonal antibody (the overall specificity of the IHC method compared with IF was 98%, and sensitivity was 87.5%).
Etta PK. Comprehensive management of the renal Transplant recipient. Indian J Transplant 2019;13:240-51.
Seemayer CA, Gaspert A, Nickeleit V, Mihatsch MJ. C4d staining of renal allograft biopsies: A comparative analysis of different staining techniques. Nephrol Dial Transplant 2007;22:568-76.
Excellent Mohamed
-Peritubular capillary complement 4d staining is one of the criteria for the diagnosis of antibody-mediated rejection, and research into this is essential to kidney allograft evaluation. The immunofluorescence technique (IF) was applied to frozen sections while the immunohistochemistry technique(IHC) was applied to paraffin-embedded tissue.
Several studies showed :
– the three-step IF method appeared to be the most sensitive and IHC the less sensitive.
-The IF method as the gold standard, found that the IHC specificity was 98% and sensitivity 87.5%. There is difficulty in interpretation of IHC, which is due to unspecific background staining.
-On average, the degree of C4d staining with an IHC method was lower by about a degree, which means that many diffusely staining cases in IF method turned focally positive in the IHC method.
– In the presence of glomerular damage strong C4d staining of the glomerular basement membrane was detected by both methods. The endothelia of arteries and arterioles were sometimes positive.
-Studies recommended the use of the IF method in frozen tissue.
IHC advantages
1- Excellent specificity.
2 – Feasible in formalin-fixed, paraffin-embedded tissue. FFPE is the usual tissue processing for kidney biopsies, so this material is always available. The IHC technique could be used when no frozen tissue is accessible.
IHC disadvantages
1. Lower sensibility. Although IHC presents an excellent specificity, it has a lower sensitivity than IF. Some false negatives were registered. The positive staining area by IHC is in some cases lower than by IF cases. Results obtained with IHC must be interpreted with some reserve since clinical relevance has only been established for IF results, and focal staining in IHC can correspond to diffuse staining in IF, for example.
2. Nonspecific staining. nephropathologists stated that IHC staining causes more difficulties in interpretation, due to the existence of unspecific staining in tubular cells and surrounding interstitium.
3 . Cost and time preparation. The IHC technique was more expensive than IF. The IHC method takes over one hour more to complete than the IF technique. The IF technique applied to frozen sections does not need previous processing and can be finished in one hour. By the IF method, the results can be available to medical staff less than two hours after tissue reception in the laboratory.
4 .Need for external controls.
–The immunofluorescence technique applied to frozen sections is the gold-standard method for complement C4d staining and the immunohistochemistry technique can safely be used when immunofluorescence is not available.
Reference:
–Ana Santos1, Helena Viana1, Maria João Galvão1, et al.C4d detection in renal allograft biopsies: immunohistochemistry vs. immunofluorescence.Port J Nephrol Hypert 2012; 26(4): 272-277
Thank You
Capillary C4d deposition has been recognized as a marker of antibody-mediated rejection (AMR). There are two methods for C4d deposition testing.
1. Immunofluorescence IF. Using monoclonal or polyclonal Ab on frozen tissue.
Advantages:
Disadvantages:
2. Immunohistochemistry IHC. Examination of formalin fixed paraffin embedded tissue
Advantages:
Disadvantages:
For the above mentioned advantages and disadvantages both tests are needed and that will increase both sensitivity and specificity. We can overcome the disadvantages of both tests by doing both of them.
Thank You
Dear All
We need to review together the sensitivity and specificity of both techniques.
Please write to us which one is more sensitive and which one is more specific.
What about the glomerular staining in IF?
IF study is more sensitive (specificity is 93% and sensitivity 94%).In the contrary,for IHC its more specific, as any positive result more than 0% considered as positive with specificity of 98% and sensitivity of 87%) formalin fixed, paraffin embeded specimen reduced sensitivity in IHC in comparison to frozen section in IF .IF would give false positive stainig in glomeruli.
Reference:
1)Lecture of prof .Halawa
2)Comparison of C4d immunostaining methods Renal allograft biopsies. Megan L et al.CJASN 2006
Thank You
IHC is more specific and less sensitive
IF is more sensitive but less specific
Thank You
The test with higher sensitivity is IF, while IHC carries a higher specificity.
Glomerular IF staining is used as a control in IF method.
Glomerular mesangial staining was shown in frozen sections while in paraffin-embedded tissues it was along the glomerular capillary wall.
Two techniques utilized for C4d identification include immunofluorescence (IF) and immunohistochemistry (IHC). Both the techniques have their own advantages and limitations.
A) Immunoflourescence (IF): C4d staining in IF has following features – (1,2)
B) Immunohistochemistry (IHC): C4d staining in IHC has following features – (1,2)
A study compareing IF and IHC concluded that IF was better suited for diagnostic and research purposes as C4d detection by IHC was lower. (4)
Probably, this is the reason that in C4d>0 is significant in IHC whereas in IF, C4d 2or 3 is deemed significant.
Considering all the pros and cons, it would be best to have a combination of both the techniques. But If only one needs to be chosen, IHC would be better due to no need of extra tissue (formalin fixed sample can be used).
References:
1) 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.
2) Khairwa A. The relevance of complement C4d staining in renal allograft biopsies. Indian J Transplant 2020;14:94-8.
3) Etta PK. C4d staining and antibody-mediated rejection in renal transplantation: Current status. Indian J Transplant 2020;14:197-201.
4) Seemayer CA, Gaspert A, Nickeleit V, Mihatsch MJ. C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques. Nephrol Dial Transplant. 2007 Feb;22(2):568-76. doi: 10.1093/ndt/gfl594. Epub 2006 Dec 12. PMID: 17164320.
It is a piece of art
Which one is more sensitive Amit?
Thank you Sir.
Immunofluorescence is more sensitive.
No it is not needed
There are advantages to both methods for detection of C4d – for example, it is recognized that detection of C4d by immunofluorescence provides the most sensitive and specific marker for C4d positivity . However, immunofluorescence studies require additional tissue, which may not be practical for protocol biopsies or for retrospective studies. Meanwhile, there is considerable variation between centers on thresholds for “focal” or “diffuse” positive staining, both by immunofluorescence and by immunoperoxidase, rendering it difficult to compare results for C4d positivity between centers. (1)
Ludovico-Martins H .et al demonstrated that frozen-IHC and paraffin-IF can be considered alternative techniques to frozen-IF for C4d detection. The paraffin-IHC technique displayed the lowest concordance rate for C4d detection.(2)
The sensitivity of IF using a monoclonal antibody to C4d in frozen sections is greater than immunoperoxidase stains using the polyclonal antibody in formalin-fixed paraffin-embedded FFPE tissue. Staining for C4d by IF was described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to the 2003 Banff conference. Medullary vessels are typically positive and can be the only place of C4d positivity in cases with marked edema and cortical injury. In IHC, strong staining is usually not seen as tissue pretreatment influences staining intensity. IHC demonstrated a substantially lower prevalence and extent of C4d deposition in PTC and had a lower reproducibility than IF.
The most sensitive method for C4d is the 3-step indirect IF on frozen sections using one of the monoclonal antibodies. However, many prefer to use the 2-step indirect IF method with the monoclonal antibody because of its simplicity, shorter time, and relatively low cost.
Although more sensitive, IF requires extra biopsy sample (other than FFPE tissue) and frozen sections facility. As IHC is feasible in FFPE tissue, it can be easily done from biopsy tissue submitted for light microscopy study when frozen section facility (for IF) is not available or when extra biopsy tissue is not available for frozen sections. (3)
Reference
1- Crary G Set al . Optimal Cut-off Point for Immunoperoxidase Detection of C4d in the Renal Allograft: Results from a Multicenter Study .Transplantation. 2010 ; 90(10): 1099–1105.
2-Ludovico-Martins H .et al .Analysis of Different Staining Techniques for C4d Detection in Renal Allograft Biopsies. Transplantation Proceedings 2009 ;41(3):862-865.
3-Kumar Etta P. C4d staining and antibody-mediated rejection in renal transplantation: Current status.Indian Journal of Transplanation 2021;14(3):197-201.
Well done Doaa
Based on what you have written, we need both of them. Do not forget, IF give positive control in the glomeruli
☆Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing?
▪︎ My answer is No
Notes:
________
C4d staining:
▪︎Currently, there are 3 commercially available antibodies for stainingof C4d. Two of these antibodies are monoclonal and are usually used with either a 3- or a 2-step indirect immunofluorescence (IF) methodology on frozen sections [1].
and a polyclonal antibody used on formalin-fixed, paraffin-embedded tissue section with an immunoperoxidase detection system [1].
☆So to answer the question Why?
▪︎All methodologies and antibodies tested provided adequate results with only minor differences between them[2], so we don’t need need both IHC and IF for C4d testing
▪︎The sensitivity of IF using a monoclonal antibody to C4d in frozen sections is greater than immunoperoxidase stains ( the most sensitive is the 3. steps IF).
▪︎The 2-step indirect IF method with the 2. steps is preferable because of its simplicity, quick turnaround time, and relatively low cost.
▪︎
▪︎In IHC, strong staining is usually not seen as tissue pretreatment influences staining intensity.
▪︎IHC demonstrated a substantially lower prevalence and extent of C4d deposition in PTC and had a lower reproducibility than IF [2].
▪︎Sometimes, the plasma in the capillaries is fixed by the formalin processing and also stains for C4d by IHC, which interferes with interpretation[2]
▪︎Nonspecific background staining is common in IHC.
▪︎ As IHC is feasible in FFPE tissue, it can be easily done from biopsy tissue submitted for light microscopy study when frozen section facility (for IF) is not available or when extra biopsy tissue is not available for frozen sections.
▪︎C4d can be detected in mesangial regions by IF (not IHC) in patients with no rejection.
______________________________
Ref
[1] Gyongyi M Nadasdy et al. “Comparative study for the detection of peritubular capillary C4d deposition in human renal allografts using different methodologies”. Hum Pathol. 2005 Nov.
[2] Praveen K.et al “C4d staining and antibody-mediated rejection in renal transplantation: Current status” Volume : 14 | Issue : 3 | Page : 197-201
Thanks
The answer is No, we don’t need to do both IHC and IFC(Troxell et al., 2006).
Immunofluorescence is still considered the gold standard.
The sensitivity and specificity of Immunoperoxidase is 87.5% and 97% respectively.
IHC has acceptable sensitivity and specificity as compared to immunofluorescence.
IF is performed on frozen sections of tissue that were transported in saline, stained with monoclonal ant-C4d antibody.
IHC performed on paraffin-embedded tissue stained with polyclonal C4d antiserum.
TROXELL, M. L., WEINTRAUB, L. A., HIGGINS, J. P. & KAMBHAM, N. 2006. Comparison of C4d Immunostaining Methods in Renal Allograft Biopsies. Clinical Journal of the American Society of Nephrology, 1, 583-591.
Thanks
1-IHC: It is possible to store pieces of paraffin-embedded tissue for further evaluation (IHC offered a good tissue sample for analysis, with good preservation of histologic features )
The method is more specific than IF, although it is less sensitive (there is no positive staining of Glomeruli) according to Banff’s score, with c4d > 0 it is statistically significant.
The process is more time-intensive (during the preparation phase) and there are issues in interpreting
Positive findings for C4d in paraffin do not rule out the possibility of positive results in frozen material.
2- IF is still the gold standard because it is based on frozen sections.
Allows for the quick and sensitive detection of C4d positive at a lower cost and with greater sensitivity.
Disadvantages
Specificity has been reduced ( False positive staining of glomeruli on the background )
C4d was found in the mesangium of the glomerulus in frozen native specimens, but not in paraffin sections that had been exposed to light for a brief period of time (and therefore could not be preserved for subsequent evaluation).
Ref: H Ludovico-Martins, C Silva et al, (Analysis of different staining techniques for c4d detection in renal allograft biopsies)
Thanks
Immunostaining of renal allograft biopsy for C4d deposition has become an important diagnostic tool in the recognition of humoral – mediated graft rejection.
Presence of C4d deposition in PTC have correlated with poor graft outcome.
Detection of C4d deposition in PTC have incorporated into Banff 97 classification system as criteria for diagnosis of humoral rejection in renal allograft.
C4d is a fragment of the classical complement pathway,C4 is activated by Ag-Ab complex.,C4 is activated and cleaved into C4a ans c4b then c4b cleavage into c4c and c4d,this fragment contain the covalent bond to the tissue and remains at the site of complement activation for longer period in contrast with the complement fragment.
tow technigues have been used to detected c4d immunostaining in the allofraft biopsy
IMMUNOFLUORESCENCE
It is gold standard method
more sensitive than IHC
staining intensity affect by length of time c4d was present at Ag/Ab reaction site
applied to frozen section of fresh tissue
positive result indicated by linear staining within preitublar capillaries of cortex and or medulla,glomerular staining is not specific ,
IMMUNOHISTOCHEMISTRY
technique applied to paraffin -embedded tissue of renal allograft biopsy.
disadvantage :
difficult in interpretation due to nonspecific staining in tubular cells and interstitium
more specificity but lower sensitivity to c4d detection in allograft dysfunction.
regarding if we need both technique,no .as we mention above the IF is first option if not available ,IHC can be used
why?because some of studies shows that all cases negative by IF also negative by IHC and all positive cases by IF are positive by IHC
Reference:
1) Immunoadsorption in severe C4d-positive acute kidney allograft rejection: a randomized controlled trial.
Böhmig GA, Wahrmann M, Regele H, Exner M, Robl B, Derfler K, Soliman T, Bauer P, Müllner M, Druml W
Am J Transplant. 2007;7(1):117. Epub 2006 Nov 15.
2) Feucht HE: Complement C4d in graft capillaries: The missing link in recognition of humoral alloreactivity. Am J Transplant3 :646– 652,2003
3) Abbas AK, Lichtman AH, Pober JS: Transplantation immunology. In: Cellular and Molecular Immunology, 4th ed., edited by Abbas AK, Lichtman AH, Pober JS, Philadelphia, W.B. Saunders Co.,2000 , pp369– 385
Thanks
Immunofluorescence (IF):
IHC:
So to increase sensitivity & specificity of C4d staining it is better to use both techniques.
References:
Excellent
In order to detect C4d in renal allograft biopsy’s two techniques have been developed these include immunfluorescence technique and immunohistochemistry , Each technique has its own pros and cons.
Immunofluorescence technique
This technique is more rapid and more sensitive and gold standard . It basically involves measurement of standing intensity which is calculated by the length of time C4d was present at antigen and antibody binding location however it is a disadvantage that samples can deteriorate quickly. This cannot be used for assessment at a later time.
Immunohistochemistry
This techniques is more specific but costly and time consuming. It is used when frozen sections facility is not available or the sample tissue is very small rendering it unsuitable for frozen section. Feasible in formalin fixed paraffin embedded tissues.
Both techniques can be used for glomerular staining however IF is more sensitive and associated with positive controls .
Referrence-
Ana Santos et al. C4d detection in renal allograft biopsies. immunohistochemistry vs immune fluorescence. Port.J Nephrol hypert.2012;24/6: 272-277.
Thanks
Renal biopsy is gold standard for diagnosis of
Acute rejection in renal transplant.
C4d Staining is a useful adjacent marker of the humoral limb of rejection boht in early
and late post transplant period.
Method : using two methods
immunohistochemistry and 1.immunofluorescencetechnique:
A.Feasble in formalin-fixed paraffin-embedded tissue.
B.The immunohistochemistry metho presents an excellent specificity but lower sensitivity to C4d detection in allograft dysfunction.
C.The evaluation is more difficult, requiring a more experienced observer .
D. Difficulties in interpretation, due to nonspecific stainingin tubular cells and surrounding interstitium.
E. cost and time preparations
F.Need for external control.
G.IHC offers several advantages. it was performed on serial levels of the same tissue therefore, it offers a generous tissue sample for analysis, with good preservation of histologic features, which is important for scoring nonatrophic areas. Exclusion of atrophic areas of cortex in scoring IF samples was significantly more difficult. Automated IHC staining of levels cut in parallel with tissue for light microscopy permits saving of labor and tissue and results in a permanent archival slide record.
.Based on these , we conclude that the immu-nohistochemistry technique can safely be used when immunofluorescence is not available
2. immunoflourecence (IF) staining need adequate fresh tissues in frozen section is more sensitive for C4D detection but can give false postive staining in glomeruli , its rapid test and should be interpreted at same time , less cost but less specific compared to IHC .
Ref:
1.C4d Detection in Renal Allograft Biopsies: Immunohistochemistry vs. ImmunofluorescenceA. Santos, H. Viana, +2 authors F. Nolasco Published 1 October 2012Biology, Medicine portuguese journal of nephrology and hypertension.
2.
Comparison of C4d Immunostaining Methods in Renal Allograft Biopsies
Megan L. Troxell, Lauren A. Weintraub, John P. Higgins and Neeraja Kambham
CJASN May 2006, 1 (3) 583-591; DOI: https://doi.org/10.2215/CJN.00900805
.
Thanks
Yes, both needed for C4D testing in ABMR and its part of daignositic banff criteria .The IHC staining usually done in formalin fixed and paraffin-embedded tissue even small biopsy or inadequate tissue can be used but need external control, more time and more cost , IHC testing very specific for C4D staining in ABMR .HC staining need experities in interpretaion due to the nonspecific staining background in the tubules and interstitium.
immunoflourecence (IF) staining need adequate fresh tissues in frozen section is more sensitive for C4D detection but can give false postive staining in glomeruli , its rapid test and should be interpreted at same time , less cost but less specific compared to IHC .
IHC can be safely used with 100% specifcity if the IF staining not available(2).
References:
1- The relevance of complement C4d staining in renal allograft biopsies
Anju Khairwa:Indian Journal of Transplantation, Year 2020, Volume 14, Issue 2 [p. 94-:98.
2-C4d Detection in Renal Allograft Biopsies: Immunohistochemistry vs. ImmunofluorescenceA. Santos, H. Viana, +2 authors F. Nolasco Published 1 October 2012Biology, Medicine portuguese journal of nephrology and hypertension.
Agree with you
Two techniques are mostly used for identification of C4d IHC and IF in allograft biopsies of the kidney .
IF technique applied to frozen sections whereas IHC technique used to paraffin embedded tissue used routinely in various laboratories .
C4d is significant staining in 1% or more of the PTC for formalin/IHC IP, or 10% or more for frozen/frequency IF.
Immunofluorescence
advantages of IHC
disadvantages of IHC:
The IHC method for C4d detection feasible in formalin fixed, paraffin embedded tissue, it is used when frozen sections facility not available, IHC also useful when small biopsy tissue or tissue not available for frozen sections .
What about glomerular staining in IF technique?
Two techniques are mostly used for identification of C4d in allograft biopsies of the kidney:
1. Indirect immunofluorescence (IF):
applied to frozen sections
In acute and chronic AMR‐positive C4d stain with IF technique is described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to a consensus at 2003 Banff Conference.
The scoring of C4d is significant by IF on frozen section ie C4d2 or C4d3
C4d is significant staining in 10% or more for frozen/frequency
C4d detection by IF is more sensitive.
Less expensive .
Rapid detection of C4d positivity.
IF method for C4d requires extra tissue and frozen sections facility.
Can not be stored for latter assessment.
Still the gold standard technique
2. Immunohistochemical (IHC):
IHC technique used to paraffin‐embedded tissue used routinely in various laboratories.
C4d staining is crisp, linear, continuous, diffuse, and lying around in the PTC wall, while the strength typically is slighter and changeable, but it may have a finely granular pattern in high power.
The scoring of C4d is significant by IHC C4d >0 on paraffin sections.
C4d detection by IHC more specific
The IHC method for C4d detection feasible in formalin‐fixed, paraffin‐embedded tissue, it is used when frozen sections facility not available, IHC also useful when small biopsy tissue or tissue not available for frozen sections.
The disadvantage of C4d staining by IHC are lower sensibility, nonspecific background staining, more costly and more time consuming, and it needs external controls.
IHC method Widely available.
Uses the same sample that is processed for LM .
Reference:
The Relevance of Complement C4d Staining in Renal Allograft Biopsies
Anju Khairwa*
Department of Pathology, ESIC Model Hospital, Gurugram, Haryana, India.
Thanks
· Methods of c4d detection:
o IF (more sensitive), but mostly requires fresh tissue in frozen section, excess tissue and must be visualized immediately after staining.
o IHC (immune-histochemistry or immune-peroxidase), can be done on paraffin sections. Can be interpreted at any time but less sensitive than IF.
o The disadvantage of IHC are lower sensitivity, nonspecific background staining, more expensive and time consuming, and it needs external controls
o C4d deposits may be linear, circumferential or crisps. It is dynamic and can vary.
o C4d in IF appearance is “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” (2003 Banff ), while in IHC, C4d staining is crisp, linear, continuous, diffuse, and lying around in the PTC wall (less strength but has granular pattern in high power).
· According Banff diagnostic criteria:
o C4d staining in PTCs by IHC on paraffin sections (at least 10% PTC positive is considered as significant) as it is less sensitive.
o In contrast, by IFs on frozen sections Banff scores C4d2 or C4d3 is considered significant (more sensitive).
Thanks
C4d deposition can be measured by
1- Immunefluresnt (IF) using monoclonal or polyclonal Ab on frozen tissue
· Advantages of IF
more sensitive than IHC ( both 2 and 3 step indirect IF on frozen section using monoclonal Ab)
and 2 step method is easier , takes shorter time and relatively lower cost
· Disadvatages
Requires extra biopsy tissue and frozen section facility
2- Immunehistochemistery (IHC) examination of formalin fixed paraffin embded tissue
Advantages of IHC
· doesn’t require extra biopsy tissue as can be done on biopsy sample prepared for LM
Disadvantages
· Less sensitive than IF
· Tissue pretreatment affects intensity of C4d staining
3 step indirect IF on frozen section is the gold standard test for detecting C4d deposits but if un available IHC has comperable sensitivity
Thanks Fatima
Expected more
Do we need both Immunoperoxidase (IHC) and
Immunofluorescence for C4d testing? Why?
Methods used for C4d detection which use :
Frozen tissue for immunofluorescence (IF) (monoclonal antibodies are more commonly used than polyclonal antibodies.)
Formalin-fixed paraffin-embedded (FFPE) tissue for
immunohistochemistry (IHC) (using polyclonal antibodies)
The sensitivity of IF using a monoclonal antibody to C4d in frozen
sections is greater than immunoperoxidase stains using the
polyclonal antibody in FFPE tissue.
Specificity of the IHC method compared with IF IS 98%.
IHC is feasible in FFPE tissue, it can be easily done from biopsy
tissue submitted for light microscopy study when frozen section
facility IS NOT AVILABLE.
Also when extra biopsy tissue is not available for frozen sections.
C4d can be detected in mesangial regions by IF (not IHC) in patients
with no rejection due to glomerular deposits seen by IF.
Agree
Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?
Ithink we need to do both
# Study condected that : normal glomeruli show mesangial C4d staining with an IF detection method applied to frozen tissue but not with the polyclonal antibody applied to formalin fixed, paraffin embedded tissue (IHC method)
#The sensitivity and the specificity of the IHC method is acceptable in comparison with the IF method.
# Although at the present time IF is considered the gold standard IF/ IHC discordant cases were seen in both no treatment and aggressive therapy groups the follow up graft function in these cases does not show any trend to suggest superiority of positive IF or IHC staining.
# In renal biopsy practice, IHC offers several advantages it was performed on serial levels of the same tissue that was cut for light microscopy, therefore IHC offered a generous tissue sample for analysis with good preservation of histologic features which is important for scoring nonatrophic areas.
# Exclusion of atrophic areas of cortex in scoring IF samples was significantly more difficult.
# So careful correlation of C4d results, donor-specific serum testing, and histologic features should be performed in each case and further correlated with clinical history.
References
Abbas AK, Lichtman AH, Pober JS: Transplantation immunology. In: Cellular and Molecular Immunology, 4th ed., edited by Abbas AK, Lichtman AH, Pober JS, Philadelphia, W.B. Saunders Co.,2000 , pp369– 385Google Scholar
Mauiyyedi S, Colvin RB: Humoral rejection in kidney transplantation: New concepts in diagnosis and treatment. Curr Opin Nephrol Hypertens11 :609– 618,2002
CrossRefPubMedGoogle Scholar
Bohmig GA, Exner M, Watschinger B, et.al Regele H: Acute humoral renal allograft rejection. Curr Opin Urol12 :95– 99,2002
Agree
4. Do we need both Immunoperoxidase (IHC) and Immunofluorescence for C4d testing? Why?
Each one has its own pros & cons
1. Indirect immunofluorescence (IF):
– Uses monoclonal anti-C4d antibodies on frozen tissue.
– Relatively simple, highly sensitive, & specific
– Disadvantages include:
i. Requires a frozen section
ii. Requires a sample different from the one used for LM, thus less satisfactory correlation.
iii. Requires fluorescent microscope.
2. Immunohistochemical (IHC):
– Uses formalin-fixed paraffin-embedded sections with polyclonal anti-C4d antibodies.
– Widely available.
– Uses the same sample that is processed for LM so no need for a second core.
– Several studies showed high sensitivity & specificity
– Disadvantage: leads to a loss of sensitivity of C4d of about 30%, compared to IF on frozen tissue.
References
1. Bowdler AL, Griffiths DF, Newman GR. The morphological and immunohistochemical analysis of renal biopsies by light and electron microscopy using a single processing method. Histochem J 1989;21:393-402.
2. Troxell ML, Weintraub LA, Higgins JP, Kambham N. Comparison of C4d immunostaining methods in renal allograft biopsies. Clin J Am Soc Nephrol 2006;1:583-91. file:///C:/Users/TOSHIBA/AppData/Local/Temp/msohtmlclip1/01/clip_image001.gif
3. Santos A, Viana H, Galvão MJ, Carvalho F, Nolasco F. C4d detection in renal allograft biopsies: Immunohistochemistry vs. immunofluorescence. Port J Nephrol Hypertens 2012;26:272-7.
4. Mauiyyedi S, Crespo M, Collins AB et al. Acute humoral rejection in kidney transplantation: II. Morphology, immunopathology, and pathologic classification. J Am Soc Nephrol 2002; 13:779-787.
5. Bohmig GA, Exner M, Habicht A et al. Capillary C4d deposition in kidney allografts: a specific marker of alloantibody-dependent graft injury. J Am Soc Nephrol 2002; 13:1091-1099.
Excellent
Both techniques are very useful in the detection C4d with different sensitivity and specificity
First IHC :
based on Paraffin-embedded sections
can be saved for later assessment (IHC offered a good tissue sample for analysis, with good preservation of histologic features )
More specific than IF however less sensitivity (No positive staining of Glomeruli )
according to Banff’s score with c4d > 0 it’s significant
However disadvantages:
less sensitive than IF
more Expensive
More time consuming ( during Preparation ) and difficulties in interpretation
Negative findings for C4d in paraffin do not exclude positive findings in frozen material.
2- IF
Still gold Standard
based on frozen sections
Allows rapid and sensitive detection of C4d positivity
less expensive
more sensitive
Disadvantages
Less specific ( False positive staining of glomeruli on the background )
C4d was expressed in the mesangium of the glomerulus in frozen native specimen but not in paraffin sections
of short time ( can’t be stored for later assessment )
Both techniques can be used (to improve of both sensitivity and specificity )
using double staining of immunofluorescence (C4d/CD34 double-immunofluorescence ) improves the detection of C4d with increasing specificity
Ref :
C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques
Christian A. Seemayer Ariana Gaspert, Volker Nickeleit, Michael J. Mihatsch
,
C4d/CD34 double-immunofluorescence staining of renal allograft biopsies for assessing peritubular capillary C4d positivity
Kuang-Yu Jen1 , Thuy B Nguyen1 , Flavio G Vincenti 2 and Zoltan G Laszik
Well done
Nice to read your contribution