Dear All
Review the level of evidence. Follow this diagram
Radwa Ellisy
2 years ago
Please summarise this article
Kidney transplantation is the treatment of choice for ESRD patients. However, the allograft might be endangered by rejection episodes, of which ABMR is the worst. ABMR accounts for 20-48% of all rejection episodes.
According to Banff classification, ABMR could be diagnosed by evidence of tissue injury (microvascular inflammation), evidence of antibody reaction (c4d staining) and serological tests detecting DSA. However, ABMR may be diagnosed in absence of C4d staining and called c4d negative ABMR. –This is a retrospective study that included 987 transplant recipient graft biopsies between 2013 and 2015. The specimens were stained for H&E, and c4d immunohistochemistry.
The histopathological findings were defined as
1- C4d positive ABMR: glomerulitis, DSA and c4d staining score more than 0 by IHC, and more than 2 by IF
2- C4d negative ABMR: C4d <2 on IF, acute TMA in the absence of any definitive cause. they concluded:
– low incident value for c4d negative rejection.
-No specific risk factors could be detected as causes for c4d negative ABMR.
-Timing for c4d negative was comparable to that for C4d positive rejection, both early and late.C4d negative ABMR had a better outcome than positive.
What is the kind of this study?
Retrospective cohort study
What is the level of evidence?
III
Wee Leng Gan
2 years ago
This is a retrospective study from January 2013 to December 2015 in India to illustrate the incidence and outcome of C4d-negative ABMR with level 3 evidence. Allograft proven immunological rejection were included. Recipients who underwent ABO incompatible transplants and defaulted follow- up were excluded. Allograft biopsies with immunological injuries classified as Group 1: pure Antibody Mediated Rejection ( ABMR ), Group 2: combined ABMR with concurrent T-cell-mediated rejection (TCR), and Group 3: pure TCR. Groups 1 and 2 again subdivided into subgrouped as C4d positive (Group 1a and 2a) or C4d negative (Group 1b and 2b). Graft function was measured by serum creatinine. Out Of the 987 allograft biopsies, 43.3% (404) of biopsies revealed immunological injury. Of these, 27.7% of the biopsies revealed pure ABMR (Group 1), 60.6% revealed combined ABMR with TCR (Group 2), and 11.3% revealed pure TCR (Group 3). The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%. The C4d-negative rejections were 18.48% and 18.7% respectively in Group 1 and Group 2. The mean serum creatine were higher in c4d negative rejection at the end of three years follow-up. In conclusion, C4d-negative ABMR has a low incidence, presents early after transplantation but carries better outcome than C4d-positive ABMR. However, further trial with longer duration is needed.
Nandita Sugumar
2 years ago
Summary
C4d detection can be called a footprint of antibody response. Although this product does not have any functional role, it serves as evidence of humoral rejection of graft for transplant teams.
C4d forms an important part of diagnostic measures for rejection, with treatment protocols including a combination of plasmapheresis, IVIG, immunoadsorption, Tacrolimus/mycophenolate mofetil, and Rituximab.
Banff criteria for ABMR and its types are the focus of this paper.
The first type is seen in the early post transplant period, in previously sensitized patients, with higher possibility of C4d positivity.
The second type is seen in the late post transplant period and is due to de novo DSA and is likely to be C4d negative. Mean serum creatinine level can be low in this group.
C4d negative ABMR has better chances of graft survival but poor graft function reflected by higher serum creatinine level.
In conclusion, this paper relies on data post transplant to show that C4d negative ABMR is to be taken as seriously as C4d positive ABMR, and treated aggressively irrespective of absence of TCR.
Type of study : Retrospective study
Level of evidence : 3
ahmed saleeh
2 years ago
Level of evidence 3
Biopsy still remains the gold standard and provides valuable insights intothepathogenesis ofearly and late allograft injury
Alloantibodies preferentially attack the peritubular and glomerular capillaries in contrast to Tcells which characteristically infiltrate tubules and arterial endothelium
C4b/C4d remains bound in the tissuefor several daysafter Ig and C1 have been released.
Covalent binding renders C4d, a stable moleculeto be easily detected by immunohistochemistry
Detection of C4d is regarded as “footprint” of an antibody response and is currently the best single marker of complementfixing circulating antibodies to the endothelium .
is currently the best single marker of complement fixing circulating antibodies to the endothelium
The presence of DSAs within the graft does not necessarily mean activation of thecomplement, pathological changes, and graftdysfunction, since accommodation may occur
twomajorphenotypes of ABMR. The first type appears early in the posttransplant period in apresensitizedpatient and is more likely to be C4dpositive.
The second type develops late posttransplant and is due to de novo DSA development and islikely to be C4dnegative.
C4d Negative subset of patients also needs tobe treated aggressively like those with C4d-positive ABMR irrespective of concurrent or absence of TCR.
AMAL Anan
3 years ago
What is the level of evidence?
Level of evidence III
AMAL Anan
3 years ago
What is the kind of this study?
Retrospective cohort study.
AMAL Anan
3 years ago
Please summarise this article
-Renal transplantation (RT) is a well-established therapy for patients with end-stage renal disease. Renal allograft dysfunction (RAD) is a common and complex problem of RT. Appropriate management is critical for longterm graft function and survival.
– ABMR generally has a worse prog- nosis and requires a different form of therapy
than T-cell-mediated rejection (TCR).
-ABMR is caused by antibodies directed against donor-specific human leukocyte antigen (HLA) molecules or other cell antigens. The most common
mechanism underlying ABMR is an amnestic robust antibody response that originates from previous antigenic exposure or de novo development of DSA.
-C4d is a fragment of complement C4b, an activation product of the classic complement
pathway. C4b (and C4d) contains an occult sulfhydryl group that forms a covalent, thio- ester bond with nearby proteins on activation by antibody and C1. C4b/C4d remains bound in the tissue for several days after Ig and C1 have been released.
-Detection of C4d is regarded as an indirect sign, a “footprint” of an antibody response.
-Definitions Adequacy – Optimal biopsy was defined as aspecimen with at least 10 nonsclerotic glomeruli and two arteries, a marginal biopsy having seven to nine glomeruli and one artery and a minimally acceptable biopsy having seven
glomeruli and one artery. Specimens with <7 glomeruli or no arteries or with only medulla were considered as inadequate for interpretation . Histological categories were classified as per Revised Banff’13 diagnostic categories for RA biopsies.
-The incidence of C4d-negative ABMR is strikingly low in our study. The demographic
profile or clinical symptoms are not helpful in determining the risk factors for the
development of C4d-negative ABMR. In our study, C4d-positive and C4d-negative ABMR
shows similar frequencies of concurrent cell- mediated rejection and both can occur early or late posttransplant. Graft outcomes were com- paratively better in the cohort of patients who developed C4d-negative ABMR as compared to those who have C4d-positive ABMR. However, longitudinal prospective studies are required to know the clinical significance and long-term outcome of C4d-negative ABMR. Molecular studies would be helpful to deter- mine antibodies responsible for C4d-negative ABMR to design targeted therapy approach to this group so as to prevent the morbidity associated with this type of injury and impro- ving graft survival in this cohort of patients.
Nasrin Esfandiar
3 years ago
• Renal allograft biopsy is the gold standard for diagnosis allograft dysfunction. Acute rejection (AR)has several types: hyper acute, accelerate acute, acute and chronic active rejection with cellular and /or cellular and humoral immunological pathways. AR determines allograft survival and its long-term outcome. ABMR is responsible for 20-48% of ARs and has worse prognosis and requires specific treatment. Donor specific antibodies (DSAs)can be performed before TX, or be de novo which are produced after TX and discovered 1960s and 1970s respectively. DSA can be anti-HLA class I and II Ags or against non-HLA Ags such as M1CA, M1CB. C4d has a covalent binding to endothelial cell surfaces and remain near the sites of C4 activation B and easily detected by IHC foot print of complement fixing Ab response and endothelial injury. High-titer DSAS cause ABMR and low titer can develop accommodation.
• This retrospective study reviewed allograft biopsies to find incidence of C4d-negative ABMR and compare their outcome with C4d positive ones. Renal biopsy was reported by five pathologists and a final report was generated. Variables such as age, gender, indication of biopsy and serum creatinine were collected. Biopsy specimens were assessed by light microscopy and C4d IHC. Luminex was performed and MFI>1000 was considered positive. Three group of biopsy were identified: 1-ABMR 2- ABMR+ ACR 3- ACR (a)C4d+ and (b)C4d- were subgroup for group 1 & 2. Patients were treated according their biopsy results.
TCR received methylprednisolone 3-5 days and then ATG if resistant.
ABMR(C4d+): plasmapheresis (40 mg/kg /session) and 5 gr IVIG (alternate days) then bortezomib (1.3 mg/m2) on days 1,4,7,11 or rituximab.
Combined TCR+ABMR: methylprednisolone and ATG then plasmapheresis.
Outcome was determined by graft loss.
• Results: of 934 allograft biopsies, 404 cases showed injury. ABMR was shown in 27.7% of biopsies (group D). Mixed ABMR +TCR (group 2) in 60.6% and pure TCR in 11.3% of cases. C4d negative rejection was shown in 18.48% and 18.7% in group 1& 2.
• The incidence of C4d negative (group1b) was 4.7% in males and 0-4.9 % in females and for group2b was 1.98% in females and 9.4% in males. There was more C4d +rejection comparing to C4d – ABMR in this study. DSA was detected in all patients with ABMR and none of TCR cases. Better HLA matching was associated with better graft survival. Graft survival was better in C4d –group comparing C4d+ group, although mean Scr was higher in this group. There was no graft loss in pure C4d- ABMR group. They concluded that C4d- ABMR has low incidence, early presentation and better outcome than C4d –positive AMBR. Pre sensitized patients were more likely C4d+ where as those who developed donor DSA were more likely C4d-.
• This is a retrospective study with level of Evidence of 3.
ABM rejections occur in 5-7 % of renal transplants and 20 % -48 % of acute rejections
usually may be due to Pre formed DSA, or Denovo DSA against Class I, II HLA Antigen
or antibodies against the non-HLA antigen ( MICA, MICB antigens, platelet-specific antigens ACE pathway and chemokines and its receptors )
C4d positive biopsies associated with poor prognosis and is considered an excellent marker for the presence of antibodies directed against allograft kidneys.
C4d positive ABM rejection is defined as :
presence of :
– DSA
-Glomerulitis and peritubular capillaritis
– C4d positive ≥ 2 in IF staining or >0 with IHC staining
C4d negative ABM rejection defined as :
presence of :
-DSA
–Glomerulitis peritubular capillaritis (g >0 and PTC >0 or g +PTC ≥ 2 or g>0 or PTC>0 )in addition to TMA with absence of etiology)
C4d positive <2 in IF staining or =0 with IHC staining Study Design :
All biopsies were compared with follow-up of creatinine in patients
and divided into 3 groups :
GroupI :ABM rejection
Group II : ACR + ABM rejections
Group III: ACR
with groups, I and II subdivided into subgroups C4d positive or C4d negative Rejections treatment:
ACR : methyl prednisolone 500 mg/day for 3–5 days + ATG for resistant cases with dose 1.5 mg/Kg
C4d positive ABMR: Plasma exchange 3sessions + IVIG 5 gm on alternate days + either
bortezomib with dose of 1.3 mg/meter square for 1,4, 7 , 11 days
or Rituximab 500 mg single dose
Combined ACR + ABM : methyl prednisolone + ATG + plasma exchange
Outcome :
Biopsies of pure ABMR: 27.7 %
Biopsies of Mixed rejections : 60.6 %
Biopsies of Pure ACR: 11.3 %
C4d negative 18.4 in group I ,18.7 % in group II
with graft survival better in the C4d negative group compared to the C4d positive group Limitations :
No rebiopsy was done during the follow up with the possibility of conversion of C4d negative to positive
No quantitative DSA done Discussion: ABMR of two phenotypes : Phenotype I early post-transplant in pre sensitized patient Phenotype II late post-transplant : due to De novo DSA
C4d negative ABMR tends to be late posttransplant with better graft survival than C4d positive
Endothelial injury expression is usually higher in ABM rejections
C4d negative less frequently than c4d positive Conclusion :
C4d positive and C4d negative have the same frequencies with cell-mediated rejections with poorer graft survival and can occur early or late.
Graft survival is better in C4d negative ABMR than in C4d positive ABMR What is the kind of this study?
Retrospective Study. What is the level of evidence?
Level III
Bank Muscat
3 years ago
Please summarise this article.
Acute rejection (AR) episodes are a major determinant of renal allograft survival and long term outcome of renal transplant (RT). Acute antibody–mediated rejection (ABMR) occurs in 5%–7% of all RTs and is responsible for 20%–48% of AR episodes. The prevalence of chronic ABMR
varies from 5% at one year to 20% at five years. ABMR generally has a worse prognosis and requires a different form of therapy than T-cell-mediated rejection (TCR).
Antibodies directed against donor-specific human leukocyte antigen (HLA) molecules or other cell antigens. The most common mechanism underlying ABMR.
peritubular capillary (PTC) C4d deposition in RT biopsies is strongly associated with a poor prognosis and raises the possibility that antibodies are responsible for graft loss.
C4d is a fragment of complement C4b, an activation product of the classic complement pathway.
Detection of C4d is regarded as an indirect sign, a “footprint” of an antibody response. ABMR presents most of the time as severe allograft dysfunction and its prompt diagnosis and optimal
treatment are essential.
Materials and Methods
an Institutional Review Board approved retrospective study of indicated diagnostic RABs performed from January 2013 to December 2015.
A panel of five pathologists independently reported.
The demographic data regarding patient age, gender, indication for RA biopsy, and serum
creatinine were collected. Graft function was measured by SCr levels (mg/dL).
Formalin-fixed biopsy specimens were processed for light microscopy and C4d immunohistochemistry as per manufacturer’s protocol.
For light microscopy, 3 μm thick sections were stained with hematoxylin and Eosin, Gomori’s
trichrome, periodic acid–Schiff, and Jones silver methenamine stains.
Inclusion and exclusion criteria
Patients whose biopsies revealed immunological rejection were included in the study.
Patients who underwent ABO incompatible transplants and those who were lost for follow up were excluded from the study.
Reporting of renal allograft biopsy Definitions:
Adequacy – Optimal biopsy was defined as a specimen with at least 10 non-sclerotic glomeruli and two arteries, a marginal biopsy having seven to nine glomeruli and one artery and a
minimally acceptable biopsy having seven glomeruli and one artery.
Histological categories were classified as per Revised Banff’13 diagnostic categories for RA biopsies.
Definition of C4d-positive ABMR – ABMR was defined if there was the presence of
circulating DSA along with glomerulitis and peritubular capillaritis on histopathology with
the presence of C4d deposition across the PTC membranes with an intensity of ≥2+ on
immunofluorescence and C4d >0 by immunohistochemistry on paraffin-embedded sections.
Acute thrombotic microangiopathy (TMA) in the absence of any other cause was also reported
as ABMR
Glomerulitis (g>0) – Glomerulitis was defined as the percentage of glomeruli that revealed infiltration by leukocytes in the capillaries.
Score g1, g2, and g3 were offered if <25%, 26%–50%, and >50% of glomeruli showed leukocytic infiltration, respectively.
Peritubular capillaritis – The percentage of PTCs in the renal cortex that contained neutrophils or mononuclear cells was defined as peritubular capillaritis. A score of ptc1 was offered if <10% PTCs revealed cells, ptc2 when 10% of PTCs were infiltrated by <5 cells, and ptc3 if more than 10% of PTCs revealed >10 cells/PTC
C4d scoring – Demonstration of PTC C4d deposition by immunohistochemistry was considered positive if grade was >C4d0. Score of C4d1, C4d2, and C4d3 was given if 1–9%, 10%–50%, and >50% PTCs were involved, respectively.
C4dnegative ABMR was defined as biopsies with C4d <2 on immunofluorescence or C4d >0 on
IHC, ptc> 0 and g >0, g+ptc ≥2, or g>0 or ptc >0 and acute TMA in the absence of any other cause of TMA.
Definition of TCR – TCR was defined as biopsies with tubulitis (t>1) along with vasculitis (v>0) and/or interstitial inflammation (i>2).
Study design
All the biopsies were compared for mean post-transplant and mean follow-up time period in months.
SCr levels (mg/dL) at the time of biopsy and at last follow-up were compared.
Patient and graft loss data along with SCr levels were used to determine patient and graft survival.
The biopsies were categorized into three main groups.
Group 1 comprised of biopsies that revealed ABMR,
Group 2 combined ACR +ABMR,
and Group 3 with ACR.
Group 1 and Group 2 were further subdivided on the basis of presence
(C4d positive) and absence (C4d negative) of C4d deposits on PTC membranes.
Anti-rejection regimen
All the patients who developed rejection were subjected to antirejection regimen which was decided according to the type of rejection reported on biopsies.
Patients who developed cellular rejection were treated with intravenous methyl prednisolone 500 mg/day for 3–5 days; rabbit anti-thymoglobulin (r-ATG) 1.5 mg/kg BW was added for resistant cases.
C4d-positive ABMR was treated with three sessions of plasmapheresis (with 40 mL/kg BW/session) followed by i.v. Ig 5 g on alternate days and subsequent bortezomib, 1.3 mg/m2 on days 1, 4, 7. and 11 or rituximab, 50–300 mg single dose.
For combined TCR + ABMR, methylprednisolone and rATG (in doses mentioned above) were administered followed by plasmapheresis.
Patients with C4d-negative rejection were offered monotherapy with either bortezomib or rituximab.
None of these patients underwent therapeutic plasma exchange. Outcome The death-censored graft loss was determined by the time between the date of transplantation and either date of graft failure indicated by need for dialysis, date of death, or last date of
follow-up with a functioning graft.
Patient death or dialysis dependency was the proposed end points of this study. We compared the death censored graft loss against each subgroup.
Conclusion The incidence of C4d-negative ABMR is strikingly low in this study.
What is the kind of this study? · Cohort retrospective study · What is the level of evidence? · Level 3.
Last edited 3 years ago by MICHAEL Farag
Abdullah Raoof
3 years ago
1- Summarize tis article
Alloantibodies are now considered important cause of acute and chronic rejection, differing in pathogenesis from TCR.
Alloantibodies preferentially attack a different “location,” namely the peritubular and glomerular capillaries in contrast to T cells which characteristically infiltrate tubules and arterial endothelium.
ABMR is caused by antibodies directed against (HLA) molecules or other cell antigens.
Antibodies can be directed against other donor-specific antigens such as
· MHC Class Irelated chain A antigens,
· MHC Class I-related chain B antigens,
· platelet-specific antigens,
· molecules of the renin–angiotensin pathway,and
· polymorphisms involving chemokines and their receptors.
C4d is fragment of C4 complement .It is produced by activation of classical complement pathway. C4d is a footprint of antibody response .
C4d deposition is strongly associated with circulating antibody to donor HLA Class I/II antigens and is cur rently the best single marker of complement fixing circulating antibodies to the endothe lium.
staining of allograft biopsies for the fragment C4d is a specific and reliable method for identifying lesions due to allo-antibodies against HLA Class I and/or Class II antigens.
Nearly 20%–30% of all Rejection episodes have a humoral component.
C4d staining of graft biopsies has emerged as an important tool for the diagnosis
of antibody-dependent allograft injury independent from its occurrence early or late after transplantation.
Antibodies can mediate endothelial injury through complement-dependent and independent mechanisms.
High-titer DSAs may cause ABMR, while recipients with low-titer DSAs can develop accommodation.
The presence of DSAs within the graft does not necessarily mean activation of the complement, pathological changes, and graft dysfunction, since accommodation may occur
In this study the incidence of C4d-negative ABMR was less than that of C4d-positive ABMR.
The most recent Banff meeting update highlights two major phenotypes of ABMR.
The first type appears early in the posttransplant period in a presensitized patient and is more likely to be C4d positive.
The second type develops late posttransplant and is due to de novo DSA development and is likely to be C4d negative. The second phenotype is an important factor in late graft loss.
patients.
Class II HLA molecules may be responsible and that much of the endothelial damage is mediated by NK cells and, monocytes and neutrophils; antibody-dependent cell-mediated cytooxicity.
C4d-negative ABMR usually occurs >12 months after transplantation but can occur acutely in highly sensitized patients with persistent DSAs .
Causes of negative C4d negative .
1- Technical issues related to type of fixative used,
2- different methods of C4d detection,
3- poor complement fixation by some DSAs,
4- Comple ment- independent pathway of ABMR
Studies have concluded that C4d negative ABMR tends to occur slightly later posttransplant and has a subclinical presen tation with better graft outcome than C4d positive ABMR.
Graft outcomes were comparatively better in patients whith C4d-negative ABMR compared to C4d-positive ABMR
2-What is the kind of this study?
This is retrospective study
3-What is the level of evidence?
Level 3
Zahid Nabi
3 years ago
Banff’13 update included C4d-antibody-mediated rejection (ABMR) as a separate entity responsible for graft dysfunction with limited clinical/prognostic implications.
This was a retrospective study which reviewed Renal allograft biopsies done between jan2013 till jan2016. A panel of five pathologists independently reported and consensus diag- nosis generated was finally reported.
Patients whose biopsies revealed immuno- logical rejection were included in the study. Patients who underwent ABO incompatible transplants and those who were lost for follow- up were excluded from the study.
Study design
All the biopsies were compared for mean posttransplant and mean follow-up time period in months. SCr levels (mg/dL) at the time of biopsy and at last follow-up were compared. Patient and graft loss data along with SCr levels were used to determine patient and graft survival. After analyzing the light microscopic features, the biopsies were categorized into three main groups. Group 1 comprised of biopsies that revealed ABMR, Group 2 combined ACR +ABMR, and Group 3 with ACR. Group 1 and Group 2 were further subdivided on the basis of presence (C4d positive) and absence (C4d negative) of C4d deposits on PTC membranes. Group 1a and Group 2a included biopsies with PTCs that demonstrated C4d deposition (C4d posi- tive) and Group 1b and Group 2b included biopsies that revealed the absence of C4d (C4d negative) on PTC membranes.
Anti-rejection regimen
All the patients who developed rejection
were subjected to antirejection regimen which
was decided according to the type of rejection
reported on biopsies. Patients who developed
cellular rejection were treated with intra-
venous methyl prednisolone 500 mg/day for
3–5 days; rabbit anti-thymoglobulin (r-ATG)
1.5 mg/kg BW was added for resistant cases.
C4d-positive ABMR was treated with three
sessions of plasmapheresis (with 40 mL/kg
BW/session) followed by i.v. Ig 5 g on alter-
nate days and subsequent bortezomib, 1.3
2
mg/m on days 1, 4, 7. and 11 or rituximab,
50–300 mg single dose. For combined TCR + ABMR, methylprednisolone and rATG (in doses mentioned above) were administered followed by plasmapheresis. Patients with C4d-negative rejection were offered mono- therapy with either bortezomib or rituximab. None of these patients underwent therapeutic plasma exchange.
Outcome
The death-censored graft loss was determined by the time between the date of transplantation and either date of graft failure indicated by need for dialysis, date of death, or last date of follow-up with a functioning graft. Patient death or dialysis dependency was the proposed end points of this study. We compared the death- censored graft loss against each subgroup.
A total of 987 RA biopsies from 987 patients
43
were analyzed; 5.3% biopsies were excluded from the study. Of the remaining 934 allograft biopsies, 43.3% (404) biopsies revealed immunological injury on histology. Out of these, 27.7% biopsies revealed pure ABMR (Group 1), 60.6% mixed ABMR + TCR (Group 2), and 11.3% pure TCR (Group 3). The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%. The incidence of C4d-negative rejections was 18.48% and 18.7% in Group 1 and Group 2, respectively. The incidence of C4d-negative ABMR was less than that of C4d-positive ABMR.
The incidence of C4d-negative ABMR is strikingly low in this study. The demographic profile or clinical symptoms are not helpful in determining the risk factors for the development of C4d-negative ABMR. In this study, C4d-positive and C4d-negative ABMR shows similar frequencies of concurrent cell- mediated rejection and both can occur early or late posttransplant. Graft outcomes were comparatively better in the cohort of patients who developed C4d-negative ABMR as compared to those who have C4d-positive ABMR.
amiri elaf
3 years ago
I. C4d-Negative Antibody-Mediated Rejection: A Pathologist’s Perspective and Clinical Outcome
# Please summarise this article
# Introduction
* Renal transplantation (RT) is best solution for patients with end stage renal disease.
* Renal allograft rejection is major problem of RT.
*Types of rejection that can occur including hyperacute, accelerated acute, acute, and chronic active rejections.
*The immunologic pathways involved in rejection divided into cellular (T cell mediated) and humoral (antibody/B cell mediated) pathways.
* There are a lot of diagnostic methodologies for diagnosis of rejection but biopsy still remains the gold standard method for detection early and late allograft injury.
*ABMR generally has a worse prognosis and requires a different form of therapy than (TCR).
*Alloantibodies are important mediators of acute and chronic rejection it attack the peritubular and glomerular capillaries, while Tcells infiltrate tubules and arterial endothelium.
*ABMR is caused by antibodies directed against donor-specific human leukocyte antigen (HLA) molecules or other cell antigens that originates from previous antigenic exposure or de novo development of DSA.
* Study showed that peritubular capillary (PTC) C4d deposition in RT biopsies is strongly associated with a poor prognosis and raises the possibility that antibodies are responsible for graft loss.
* Feucht et al suggested that capillary C4d deposition was an evidence for humoral alloreactivity against the graft and strongly associated with circulating antibody to donor HLA Class I/II antigens and best single marker of complement fixing circulating antibodies to the endothelium.
# Materials and Methods
This was retrospective study performed from January 2013 to December 2015.
*Data (patient age, gender, indication for RA biopsy, and serum creatinine were collected)
*Formalin-fixed biopsy specimens were processed for light microscopy and C4d immunohistochemistry
*Inclusion and exclusion criteria Patients with biopsies revealed immunological rejection were included in the study.
Patients with ABO incompatible or were lost for follow up excluded from the study.
* C4d positive ABMR was defined if there was the presence of circulating DSA along with glomerulitis and peritubular capillaritis on histopathology with the presence of C4d deposition across the PTC membranes with an intensity of ≥2+ on immunofluorescence and C4d >0 by immunohistochemistry on paraffin-embedded sections.
* C4dnegative ABMR was defined as biopsies with C4d 0 on IHC, ptc> 0 and g >0, g+ptc ≥2, or g>0 or ptc >0
* TCR was defined as biopsies with tubulitis (t>1) along with vasculitis (v>0) and/or interstitial inflammation (i>2).
*Definition of ABMR with TCR biopsies
with C4d-negative ABMR which also had
histological evidence of TCR also had g ≥1
*Detection of the DSA by si ngle antigen bead for HLA Class I and II
any normalized MFI value over 1000 was considered positive.
# Study design
*After analyzing the light microscopic features, the biopsies were categorized into three main groups. Group 1 revealed ABMR,
Group 2 combined ACR +ABMR and Group 3 with ACR.
* Group 1 and Group 2 were subdivided to Group 1a and 2a included biopsies with PTCs that demonstrated C4d deposition (C4d positive) and Group 1b and 2b included biopsies that revealed the absence of C4d (C4d negative) on PTC membranes.
# Anti-rejection regimen
Patients who developed cellular rejection were treated with IV methyl prednisolone 500 mg/day for (3–5 days) and r-ATG 1.5 mg/kg BW was added for resistant cases.
C4d-positive ABMR was treated with 3 sessions of plasmapheresis followed by i.v. Ig 5 g on alternate days and subsequent bortezomib, 1.3 mg/m2 on days 1, 4, 7. and 11 or rituximab 50–300 mg single dose.
For combined TCR + ABMR, methylprednisolone and rATG followed by plasmapheresis.
Patients with C4d-negative rejection with bortezomib or rituximab.
# Results
*Out of 934 allograft biopsies, 43.3% of biopsies revealed immunological injury on histology.
*27.7% revealed pure ABMR (Group 1).
*60.6% mixed ABMR + TCR (Group 2).
*11.3% pure TCR (Group 3).
* The overall incidence of ABMR (pure ABMR+ ABMR with TCR) was 36.27%.
* The incidence of C4d-negative rejections was 18.48% and 18.7% in Group 1 and Group 2, respectively.
*The incidence of C4d-negative ABMR was less than that of C4d-positive ABMR.
# Discussion
*The most recent Banff update highlights two phenotypes of ABMR. The first type appears early in the posttransplant in a presensitized patient and it is C4d positive the second type develops late posttransplant and is due to de novo DSA development and it is C4d negative.
*It appears that Class II HLA molecules may be responsible of the endothelial damage is mediated by NK cells, monocytes and neutrophils; antibody-dependent cell-mediated cytooxicity.
* C4d-negative ABMR usually occurs >12 months after transplantation but can occur acutely in highly sensitized patients with persistent DSAs
*Technical issues related to some causes of C4d negativity. *Autoantibodies to the angiotensin II type 1 receptor also
associated with graft loss and fibrinoid
necrosis similar to ABMR except for the absence of
PTC C4d.
* Fc receptors on NK cells (FcRIIA) also play a role in AR.
*There is significant proportion in ABMR transit between negative C4d staining and positive C4d deposition in the peritubular capillaries.
*C4d negative ABMR occur later posttransplant and has a subclinical presentation with better graft outcome than C4dpositive ABMR.
*Sis et al reported that expression of endothelial cellassociated
transcripts (ENDATs) is present in all types of rejection but higher in ABMR.
*When C4d negative ABMR occurs alone it presents later than C4d-positive ABMR but if concurrent TCR is also seen, it presents earlier than C4d-positive ABMR.
*Study concluded that C4d-positive and C4d-negative ABMR show similar frequencies of concurrent cell mediated rejection and both can occur early or late posttransplant.
*The mean SCr levels at the time of presentation was low in the C4d-negative ABMR, but the mean SCr over the mean follow-up period appears to be higher inC4d-negative ABMR compared to other groups these may be attributed to not subjecting these patients to aggressive management.
*Although the C4d negative ABMR associated with better graft survival,
it is poorer graft function reflected by the higher serum creatinine indicating the immune injury so this patients needs to be treated aggressively like those with C4d-positive ABMR irrespective of concurrent or absence of TCR.
# What is the kind of this study?
Retrospective cohort study
#What is the level of evidence?
Level of evidence is level 3
Esmat MD
3 years ago
This study is retrospective observational study with level 3 of evidence.
This study evaluates incidence and outcomes of C4d negative AMR in 987 kidney transplant recipients.
Renal allograft dysfunction can be result from ABMR and/or TCMR. Based on this article, several types of rejection including hyperacute, accelerated acute, acute, and chronic active rejection have been described. Biopsy is gold standard for evaluation of kidney dysfunction and allograft rejection in kidney transplant recipients.
Acute rejection has a deleterious effect on graft survival and ABMR is responsible for 20-48% of AR. Incidence chronic ABMR is reported between 5 -20%. ABMR has worse prognosis compared to TCMR. Formation of de novo DSA has detrimental effect on graft survival.
Alloantibodies preferentially attack peritubular and glomerular capillaries, but T cells typically infiltrate tubules and arterial endothelium.
The most common mechanisms for ABMR is amnestic robust response to previously exposed antigens and de novo DSA formation. Antibodies direct against HLA class I and class II, MICA, MICB, PLT-specific antigens, molecules of renin-angiotensin pathway, and chemokines and its receptors.
C4d is a fragment of C4b and is marker of activation of classical pathway and it remains on the endothelial cell surfaces, vascular basement membrane, and in intracytoplasmic vacuoles of endothelial cells. C4d positive staining is footprint of antibody response. C4d staining is the best marker of complement fixing antibodies to the endothelium and is an important tool for the diagnosis antibody mediated graft injury.
By upregulation of complement regulatory and inhibitory pathways, under certain conditions, antibodies can lead to graft accommodation. Low titer of DSAs can induce accommodation. Thus, presence of DSAs do not always mean activation of complement and graft injury.
Early diagnosis of ABMR is crucial because it needs heavy treatment with IVIG, plasmapheresis, immunoadsorption, and Rituximab. Two different phenotypes of ABMR were classified in Banff 2013. The first type occurs early post-transplant and in pre-sensitized patients, and more likely to be C4d positive. The second type is due to de novo DSA and present late post-transplant and more likely to be C4d negative. It is important in late graft loss, HLA class II molecules are more probably responsible, and it is more plausibly due to NK cell mediated endothelial injury. C4d negative ABMR usually occurs after 1 year post transplantation but can present in highly sensitized recipients.
Different fixation techniques, different methods of DSA detection, DSA with poor complement fixation or complement independent pathways such as anti-angiotensin II type I antibodies are causes of C4 negative staining. The FcRIIA on NK cells mediated graft injury can be another cause of C4d negativity.
Transition between C4d positive staining and C4d negative staining in ABMR is reported. C4d negative ABMR occur later post-transplant with subclinical presentation and better graft outcomes compared to C4 positive ABMR. Although ENDATs is present in all types of rejection, it presents higher in ABMR especially in C4d negative ABMR.
One study reported C4d positive and C4d negative ABMR had similar frequency of concurrent TCMR.
In this study, the overall incidence of C4d negative ABMR was 18.2 %, significantly less than C4d positive ABMR. It was correlated with later presentation but in concurrence with TCMR occurred earlier.
In C4d negative group, although serum Cr level was lower in the beginning of study, it was higher at the end of mean follow up period. The overall graft survival was 97.5% and 95.5% in C4d negative and C4d positive, respectively.
C4d negative ABMR should be treated similar to C4d positive ABMR with no hesitation.
Mohamed Ghanem
3 years ago
Introduction Types of Renal allograft rejection: Hyperacute, acute, and chronic by two immunological pathways humoral and cellular.
ABM rejections occur in 5-7 % of renal transplants and 20 % -48 % of acute rejections
usually, alloantibodies directed against peritubular and glomerular capillaries
etiology of ABM rejections: Presenistised DSA, or Denovo DSA against Class I, II HLA Antigen
or antibodies against the non-HLA antigen ( MICA, MICB antigens, platelet-specific antigens ACE pathway and chemokines and its receptors )
The poor prognosis of ABM rejections especially if associated with C4d positive biopsies
C4d has no functional role but it is considered to be an excellent marker for the presence of antibodies directed against allograft kidneys.
with an endothelial injury can be mediated either complement-dependent or not.
higher DSA leads to ABM and lower DSA leads to accommodation.
C4d positive ABM rejection is defined as :
presence of circulating DSA + glomerulitis and peritubular capillaritis + C4d positive ≥ 2 in IF staining or >0 with IHC staining
C4d negative ABM rejection defined as :
presence of circulating DSA + (g >0 and PTC >0 or g +PTC ≥ 2 or g>0 or PTC>0 in addition to TMA with absence of etiology) + C4d positive <2 in IF staining or =0 with IHC staining
Study Design :
All biopsies were compared with follow-up of creatinine in patients
and divided into 3 groups :
GroupI :ABM rejection
Group II : ACR + ABM rejections
Group III: ACR
with groups, I and II subdivided into subgroups C4d positive or C4d negative
Rejections treatment:
ACR : methyl prednisolone 500 mg/day for 3–5 days + ATG for resistant cases with dose 1.5 mg/Kg
C4d positive ABMR: Plasma exchange 3sessions + IVIG 5 gm on alternate days + either
bortezomib with dose of 1.3 mg/meter square for 1,4, 7 , 11 days
or Rituximab 500 mg single dose
Combined ACR + ABM : methyl prednisolone + ATG + plasma exchange
Outcome :
Biopsies of pure ABMR: 27.7 %
Biopsies of Mixed rejections : 60.6 %
Biopsies of Pure ACR: 11.3 %
C4d negative 18.4 in group I ,18.7 % in group II
with graft survival better in the C4d negative group compared to the C4d positive group
Limitations :
No rebiopsy was done during the follow up with the possibility of conversion of C4d negative to positive
No quantitative DSA done
Discussion: ABMR of two phenotypes : Phenotype I early post-transplant in pre sensitized patient Phenotype II late post-transplant : due to De novo DSA
C4d negative ABMR tends to be late posttransplant with better graft survival than C4d positive
Endothelial injury expression is usually higher in ABM rejections
C4d negative less frequently than c4d positive
Conclusion :
C4d positive and C4d negative have the same frequencies with cell-mediated rejections with poorer graft survival and can occur early or late.
Graft survival is better in C4d negative ABMR than in C4d positive ABMR
What is the kind of this study?
Retrospective Study. What is the level of evidence?
Level III
Drtalib Salman
3 years ago
Please summarise this article:
Introduction
Acute antibody–mediated rejection (ABMR) occurs in 5%–7% of all RTs and is responsible for 20%–48% of AR episodes. ABMR generally has a worse prognosis and requires a different form of therapy than T-cell-mediated rejection (TCR ),C4d is a fragment of complement C4b, an activation product of the classic complement pathway.
Detection of C4d is regarded as an indirect sign, a “footprint” of an antibody response.
C4d deposition is strongly associated with circulating antibody to donor HLA Class I/II antigens and is currently the best single marker of complement fixing circulating antibody.
Materials and Methods:
This was an Institutional Review Board approved retrospective study of indicated
diagnostic performed from January 2013 to December 2015,( ABO incompatible transplants and those who were lost for follow- up were excluded from the study),
C4d- negative ABMR was defined as biopsies with C4d <2 on immunofluorescence or C4d >0 on IHC, ptc> 0 and g >0, g+ptc ≥2, or g>0 or ptc >0 and acute TMA in the absence of any other cause of TMA. Aim of study: a retrospective study to determine the incidence and outcome of C4d-negative ABMR.
Results and discussion:
A total of 987 RA biopsies from 987 patients were analyzed,the overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%. The incidence of C4d-negative rejections was 18.48% and 18.7% in Group 1 and Group 2, respectively. The incidence of C4d-negative ABMR was less than that of C4d-positive ABMR.
All the patients who revealed immunological injury were subjected to qualitative DSA estimation and we found a 100% positivity in patients having ABMR. DSA was absent in all the cases with TCR,C4d-negative injury does affect the graft function if not treated aggressively and causes graft attrition in the long-term.
The overall graft survival appears to be better in the C4d-negative group as compared to the C4d-positive ABMR group in spite of the higher mean SCr levels in this group.
The overall incidence of ABMR was higher in our study. This could be attributed to various causes such as transplantation across HLA barriers, multiple transfusions, presensitized patients, and multiple pregnancies in women apart from low hygienic conditions prevailing in this country.
The overall incidence of C4d-negative ABMR in our study was 18.2%, which is much less than C4d-positive ABMR (72.3%). C4d–negative ABMR when occurs alone, usually presents later than C4d-positive ABMR (1.51 vs. 1.32 months).
Limitations :
study cohort with C4d-negative ABMR was not re-biopsied during follow-up. Hence, the possibility of C4d negative converting to C4d positive could not be determined. we did not perform quantitative DSA estimation due to financial constraints and compare with biopsy results.
CONCLUSION:
C4d-positive and C4d-negative ABMR shows similar frequencies of concurrent cell- mediated rejection and both can occur early or late posttransplant. Graft outcomes were comparatively better in tpatients who developed C4d-negative ABMR as compared to those who have C4d-positive ABMR.
What is the kind of this study?
This is retrospective study
What is the level of evidence?
Level 3
Mahmud Islam
3 years ago
This is a retrospective study (level III)
A total of 987 renal allografts were investigated. c4d positive AMBR was considered in case of 2 or more intensity for IF and >0 for IHC in addition to the classical definition of the existence of DSA and glomerulitis and capillaritis. presence of acute TMA was considered as AMBR. histological categories were defined according to revise BANFF13. biopsies were divided in 3 groups (group one that revealed acute AMBR, group to ACR and ABMR, group 3 ACR. group 1 and 2 were subdivided into either c4d positive or negative.
27% revealed pure ABMR (group1)
60.6% had mixed ABMR and TCR (group 2)
11 % had pure TCR (group 3)
the overall incidence of AMR either pure or cıomined was 36.27 %
the incidence of C4d-negative was 18.48% and 18.7 in group1 and 2 respectively. this is less than c4d positive ABMR
a limitation of this study is suspicious incidence of c4d because c4d negatives were not rebiopsied (the probability of being positive later is not excluded). DSA levels were quantified.
Ramy Elshahat
3 years ago
retrospective study to determine the incidence and outcome of C4d-negative ABMR.
Sample size 987 renal allograft (RA) biopsies obtained from 987 RA recipients were studied from January 2013 to January 2016.
All samples were subjected to light microscopy using standard stains and C4d immunohistochemistry on paraffin sections and reported according to modified Banff’s criteria.
Adequate biopsies with immunological injuries were categorized as
· Group 1: pure ABMR
· Group 2: combined ABMR with concurrent T-cell-mediated rejection (TCR)
· Group 3: pure TCR.
· Groups 1 and 2 were further subgrouped as C4d positive (Group 1a and 2a) or C4d negative (Group 1b and 2b).
43.3% (404)biopsies revealed immunological injury. Of these,
· 27.7% of the biopsies revealed pure ABMR (Group 1),
· 60.6% revealed combined ABMR with TCR (Group 2)
· 11.3% revealed pure TCR (Group 3).
· The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%, of which C4d-negative rejections were 18.48% and 18.7% in Group 1 and Group 2
The mean SCr at the end of three years follow-up in patients with C4d-negative rejections was comparatively higher. C4d-negative ABMR, recently included in Banff’13, has a low incidence, usually presents early after transplantation but carries better outcome than C4d-positive ABMR. However, further long term studies are still required for knowing the clinical course over years.
methodology
excellent study design in which diagnostic biopsies performed from January 2013 to December 2015.
A panel of five pathologists independently reported and consensus diagnosis generated was finally reported to decrease the human error in the results.
The demographic data regarding patient age, gender, indication for RA biopsy, and serum creatinine were collected. Graft function was measured by SCr levels (mg/dL).
Formalin-fixed biopsy specimens were processed for light microscopy and C4d immuno-histochemistry as per manufacturer’s protocol. For light microscopy, 3 μm thick sections were stained with hematoxylin and Eosin, Gomori’s trichrome, periodic acid–Schiff, and Jones silver methenamine stains. The C4d staining was performed on 3 μm thick paraffin sections. Biopsies negative for C4d were re-stained to rule out analytical errors associated with staining procedure. Membranous/lupus nephropathy slides were used as positive controls. Endothelial cells lining the medium caliber blood vessels were taken as internal control.
Inclusion and exclusion criteria
· Patients whose biopsies revealed immunological rejection were included in the study.
· Patients who underwent ABO incompatible transplants and those who were lost for follow- up were excluded from the study.
Reporting of renal allograft biopsy Definitions Adequacy
· Optimal biopsy was defined as a specimen with at least 10 non sclerotic glomeruli and two arteries
· a marginal biopsy having seven to nine glomeruli and one artery
· a minimally acceptable biopsy having seven glomeruli and one artery.
Specimens with <7 glomeruli or no arteries or with only medulla were considered as inadequate for interpretation.
Definition of C4d-negative ABMR – C4d- negative ABMR was defined as biopsies with C4d <2 on immunofluorescence or C4d >0 on IHC, ptc> 0 and g >0, g+ptc ≥2, or g>0 or ptc >0 and acute TMA in the absence of any other cause of TMA.
Definition of TCR – TCR was defined as biopsies with tubulitis (t>1) along with vasculitis (v>0) and/or interstitial inflammation (i>2).
Definition of ABMR with TCR – Biopsies with C4d-negative ABMR which also had histological evidence of TCR also had g ≥1 so as to qualify the criteria of ABMR.
Quantitative estimation of DSA by single antigen (SA) testing – HLA Class I and Class II antibody specificity screening was performed with Single Antigen Beads on Luminex platform following the manufacturer’s protocol. Trimmed mean fluorescence intensity (MFI) values were obtaine the flow analyzer and normalized using the formula: [(Sample #N beads – Sample negative control (NC) beads) – (NC serum #N beads – NC serum NC beads)]. Any normalized MFI value over 1000 was considered positive.
Limitations
· cohort with C4d-negative ABMR was not re-biopsied during follow-up. Hence, the possibility of C4d negative converting to C4d positive could not be determined.
· We did not perform quantitative DSA estimation due to financial constraints and compare with biopsy results.
· the mean SCr over the mean follow-up period appears to be higher in Group 1b (C4d-negative ABMR) when compared to other groups Treatment regimens were different in both groups (c4d +ve and c4d-ve)>> c4d +ve received more aggressive treatment.
Conclusion
· The incidence of C4d-negative ABMR is low in study.
· The demographic profile or clinical symptoms are not helpful in determining the risk factors for the development of C4d-negative ABMR
· C4d-positive and C4d-negative ABMR shows similar frequencies of concurrent cell- mediated rejection and both can occur early or late posttransplant.
· Graft outcomes were comparatively better in the cohort of patients who developed C4d-negative ABMR as compared to those who have C4d-positive ABMR. However, longitudinal prospective studies are required to know the clinical significance and long-term outcome of C4d-negative ABMR.
· Molecular studies would be helpful to determine antibodies responsible for C4d-negative ABMR to design targeted therapy approach to this group so as to prevent the morbidity associated with this type of injury and improving graft survival in this cohort of patients.
type of study retrospective observetional study
level of evidence 3
Murad Hemadneh
3 years ago
Please summarise this article
Introduction: This study was done in pathology department in India in the period of January 2013 to December 2015 aiming to determine the incidence and out come of C4d negative Acute Antibody Mediated Rejection (ABMR). ABMR is an important cause of acute and chronic allograft rejection and has worse prognosis than T-cell mediated rejection (TCR).
Design: This study was conducted on 987 renal allograft biopsies which were subjected to LM and C4d IHC on paraffin sections and reported according to modified Banff’s Criteria. Serum creatinine level was done at time of biopsy and was followed afterward. All patients their biopsy showed evidence of rejection was included. ABO incompatible transplant and patient who lost follow-up were excluded. Time after transplantation ranges from 5 to 8 years. After Exclusion criteria was applied 934 allograft biopsies remained of which 404 (43.3%) showed immunological injury and was divided into three groups. Group I: Pure ABMR, Group II: Combined ABMR and TCMR and Group III: Pure TCR.
Results: Group I was 27% of biopsies, Group II 60.6% and Group III 11.3%. The overall incidence of ABMR in both group I and II was 36.27% of which about 18% in both groups were C4d negative. Mean serum creatinine in patient with C4d negative ABMR was higher compared to C4d positive at the end of three years follow-up. C4d negative ABMR has better outcomes and graft survival compared to C4d positive ABMR.
What is the kind of this study?
Retrospective Study.
What is the level of evidence?
Level III
Mohamed Essmat
3 years ago
This is a retrospective study; class 3 evidence. Summary: Renal transplantation is the modality of choice for patients with ESKD. Different immunological pathways can cause rejection as cellular or humoral. Graft biopsy is considered as the gold standard method for rejection diagnosis. Acute ABMR may occur in 5%-7% of all recipients. The study included 934 graft biopsies. All recipients with evidence of immunological rejection were included in the study. Recipients with ABO incompatible transplantation & those who lost follow-up were excluded. Adequate-optimal biopsy :at least 10 glomeruli & 2 arteries. Marginal biopsy :at least 7-9 glomeruli + 1 artery. Minimal accepted :biopsy at least 7 glomeruli + 1 artery. C4d positive ABMR means the presence of DSA with glomerulitis and peritubular capilliritis in addition to positive staining of C4d across PTC membrane. C4d negative ABMR means presence of DSA with glomerulitis and peritubular capilliritis with C4d staining <2 by IF. TCR means the presence of tubulitis, vascular inflammation with or without interstitial inflammation. All patients are treated for rejection according to the result of biopsy with pulse steroid, ATG for the TCR or PTx and IVIg followed by bortizumib & rituximab for the ABMR .
Outcome: -End point of study was patient death or dialysis dependency. C4d negative ABMR may be due to: -Technical errors -poor complement fixation by DSA -complement independent pathway ABMR -antibodies against ACEII type 1 receptors -NK Fc receptors may have a role in acute rejection with C4d negative staining. It was conclude that the ABMR with C4d negative is less frequent and associated with better prognosis than ABMR C4d positive but still carries a poor graft survival.
Ahmed Faisal
3 years ago
Retrospective study
Level of evidence 3
C4d- negative ABMR:
• is less common than C4d- positive
• occurs more late (usually after one year)
• less severe than C4d- positive
Nazik Mahmoud
3 years ago
Retrospective study
Level 3 evidence
Conducted from to 2013 to 2016
Study 987 renal allograft biopsies to determine the incidence and outcome of C4d negative ABMR
They divided the patients to 3 groups
1-pure ABMR
2- combined ABMR and TCR
3-pure TCR
Then divided group 1&2 to sub group C4d positive and C4d negative (by IP to paraffin sections) they excluded those undergo ABO incompatible Transplant (5.3%)
So the result
43% had immunological reaction,27% of them revealed pure ABMR ,60% mixed rejection and 11% pure TCR
So the incidence of C4d negative ABMR was low and the graft survival was better in these group
Ben Lomatayo
3 years ago
Introduction ;
ABMR is associated worse prognosis and it is treated differently from TCMR[7]
ABMR is caused by Ab directed against HLA antigens (class I & class II) or other cell antigens[9,10]
The presence of C4d( complement component) in ptc is associated adverse outcome[13]
C4d forms covalent bond with the tissue,became more stable and detected on IHC(Indirect sign of an anti-body response = footprint) [13]
Antibodies causes endothelial injury through complement-dependent and complement-independent mechanisms
This is retrospective study to determine the incidence & outcome of C4d-negative ABMR
Material and Methods ;
Retrospective study(n = 987) between January 2013 to December 2015
Patients are divided into 3 groups ;
Group 1 ; Pure ABMR(1a C4d positive, 1b C4d negative )
Group 2 ; Combined ABMR with concurrent TCMR(2a C4d positive, 2b C4d negative)
Group 3 ; Pure TCMR
Biopsies( formalin-fixed specimens on LM & IHC) were analysed by 5 different pathologist and they gave a consensus report based on revised Banff 13 criteria
C4d staining was performed on 3 micrometer thick paraffin sections using rabbit anti-human C4d monoclonal antibodies
C4d negative cases were re-examined to exclude technical errors
The study included all patients with immunological injury and excluded those with ABO-incompatibility and lost to follow up groups
Reporting of renal allograft biopsy ;
Definitions
Adequacy(optimal) ; At least 10 non-sclerotic gloms + 2 arteries
Marginal ; 7 to 9 gloms + 1 artery
Minimally acceptable ; 7 gloms + 1 artery
Inadequate for interpretation ; < 7 gloms + no artery or ONLY medulla
C4d-positive ABMR ; DSA + glomerulitis/peritubular capillaritis + C4d deposition in ptc ( C4d intensity > =2 on IF & > 0 by IHC)
Glomerulitis(g>0); % of glomeruli infiltrated by leukocytes( g1 <25%,g226-50%,g3 > 50%)
Peritubualr capillaritis ; % of ptc contained neutrophils or monocytes( ptc<10%, ptc 10%, ptc> 10%)
Quantitative estimation of DSA ; Single antigen assay(SA) testing
Anti-rejection therapy ;
TCMR ; 500mg IV methy pred 3-5days+/- rabbit ATG 1.5mg/kg in resistant cases
2.C4d-positive ABMR ;
3 Secession of PP
IVIG 5g
Bortezomib 1.3 mg/M2 day 1,4,7,11
Rituximab 50-300 mg single dose
3.ABMR + TCMR ;
Methyl pred
rATG
PP
4.C4d negative ABMR ;
Monotherapy ; Bortezomib or Rituximab
Outcome ;
Death-censored graft loss was compared between groups
This was assess by the time between the date of transplantation and either date of graft failure(return to dialysis), date of death, or last date of follow up with functioning graft.
Statistical analysis
Continuous variable = mean & one standard deviation
Qualitative variable = proportion
P value < 0.05 = statistically significant
Results ;
Out of 943 biopsies, 43.3% showed immune mediated injury( 5% of cases did not meet the criteria)
28% = pure ABMR (group1)
61% = ABMR + TCMR(group 2)
11% = pure TCMR
36% = overall incidence of ABMR
18% = incidence of C4d -negative(group1)
19% = incidence of C4d -negative(group2)
Incidence of C4d-negative ABMR < C4d-positive ABMR
C4d-positive ABMR had the shortest time(1.32 +/- 0.28 months) of presentation post-transplant followed by C4d-negaticve ABMR(1.33 +/- 0.15 months)
C4d-positive had serum Cr higher(2.5 +/-0.24 mg/dl) than that of C4d-negative(2.11 +/-0.32 mg/dl)
DSA positivity was 100% in those with ABMR
Mean HLA was match 2.2 in pure ABMR,and 1.5 in C4d negative ABMR
Mean Cr was higher in C4d-negatvie compared with C4d-positive
Graft survival ;
C4d-negative had better allograft survival than C4d-positive despite higher Cr.
More allograft loss in C4d-posoitive(5.2%) compared to C4d-negative ABMR(2.5%)
Discussion ;
C4d staining now became important element in the diagnosis of ABMR
C4d-ve ABMR usually present after one year post-transplant and sometimes very early in highly sensitized patients. It has sub-clinical presentation and associated with better outcome than C4d+ve ABMR[21,34-36]
Important causes of C4d -ve ABMR are ; technical error, complement-independent pathway, antibodies are unable to fix complement, auto-anti-bodies(angiotensin II type I receptor Ab), and NK FC receptor mediated injury
This study showed overall high incidence of ABMR due transplantation across HLA barriers( presensitization)
Sis et al revealed that expression of endothelial cell associated transcripts are high in any patient with rejection and but it was significant in those with AMBR
The results showed that mixed C4d-ve ABMR + TCMR occurred earlier than C4d-ve ABMR alone. This is similar to what was found by Orandi et al[21]
In one study by Loupy et al ; the prevalence of C4d -ve ABMR(49%) was higher than C4d+ve ABMR(31%)[33]
Mark Haas revealed that both C4d-ve and C4d +ve ABMR had similar rates of concurrent TCMR and both can occur in early and late post-transplant period[43]. This study demonstrated the same finding.
The mean Cr during follow up was higher in C4d-ve ABMR compared to C4d +ve ABMR. Orandi et al comfirmed the same observations[21]. The possible explanation of higher Cr in C4d-ve ABMR may be due to the facts that this groups of patients generally received less form of aggressive treatment[6,26]
The overall graft survival was superior in C4d-ve groups compared with C4d+ve groups(90% vs 78%). Despite this, C4d-ve ABMR is still associated with poor graft fucntions as indicated by higher Cr levels. C4d-ve AMBR is important cause of graft attrition overtime and lead to allograft dysfunction as observed by Sis et al, Haas,Loupy et al, and Orandi et al.
Therefore this lesion has to treated equally as C4d+ve ABMR to avoid gradual allograft attrition over long run.
Limitations ;
No second biopsy done during the follow up; the importance of this is to see whether the C4d-ve status remained the same or converted to C4d+ve status. Orandi et al demonstrated the possibility of conversion from C4d -ve to C4d+ve status.
No DSA quantification and therefore no comparison with biopsy results. This was due financial reasons
Conclusions ;
This study showed TCMR occurred concurrently with both C4d-ve & C4d+ve ABMR
Allograft outcome was better in C4d-ve group compared to C4d+ve group
C4d -ve ABMR deserve aggressive treatment the same as C4d-ve entity
More studies are needed over long the give a solid evidence in this matter of C4-ve ABMR.
C4d-Negative Antibody-Mediated Rejection: A Pathologist’s Perspective and Clinical Outcome
☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆
▪︎This was an Institutional Review Board approved retrospective study of indicated diagnostic Renal Allograft Biopsies (RABs) performed from January 2013 to December 2015.
☆Introduction:
________________
▪︎ Biopsy is the gold standard for diagnosis of renal allograft dysfunction (RAD)
▪︎Alloantibodies are important mediators of acute and chronic rejection, differing in pathogenesis or “nature,” from T.cell mediated rejection (TCR).
▪︎ Alloantibodies preferentially attack a different “location,” namely the peritubular
and glomerular capillaries in contrast to Tcells which characteristically infiltrate tubules and arterial endothelium.
▪︎ABMR is caused by antibodies directed against donor-specific HLA molecules.
▪︎AR associated with the development of de novo anti-HLA DSA in recipient’s serum is a defined clinicopathologic entity carrying a poor prognosis and
▪︎Complement-neutrophil PW is the major mechanism of antibody injury.” And neutrophils in peritubular and glomerular capillaries are strongly associated with circulating anti-donor HLA antibodies.
▪︎Peritubular capillary (PTC) C4d deposition in RT biopsies is strongly associated with a poor prognosis and raises the possibility that antibodies are responsible for graft loss.
▪︎ C4d is an activation product of the C4. C4b (and C4d) contains an occult sulfhydryl group that forms a covalent, thioester bond with nearby proteins on activation by antibody and C1. C4b/C4d remains bound in the tissue for several days after Ig and C1 have been released.
▪︎Following activation C4d bind to endothelial cell surfaces and extracellular matrix components of vascular basement membranes near the sites of C4 activation.
▪︎C4d is also found in intracytoplasmic vacuoles of endothelial cells. Covalent binding renders IH.
▪︎Detection of C4d is regarded as an indirect sign, a “footprint” of an antibody response.
▪︎ No functional role of C4d per se has been reported.
▪︎C4d deposition is strongly associated with circulating antibody to donor HLA Class I/II antigens and is currently the best single marker of complement fixing circulating antibodies to the endothelium.
▪︎Capillary C4d deposition was an evidence for humoral alloreactivity against the graft.
▪︎Staining of allograft biopsies for the fragment C4d is a specific and reliable method for identifying lesions due to allo-antibodies against HLA Class I and/or Class II antigens.
▪︎ C4d staining of RA biopsies is an important tool for the diagnosis of antibody-dependent allograft injury independent from its occurrence early or late
after transplantation.
▪︎Antibodies can mediate endothelial injury through complement-dependent and independent mechanisms.
▪︎ Under certain conditions, antibodies can lead to graft accommodation (in low titre DSA)
☆Materials and Methods:
_________________________
▪︎ This is a retrospective study which determine the incidence and outcome of C4d-negative ABMR.
▪︎ It studied 987 renal allograft (RA) biopsies obtained from Jan 2013 to Jan 2016 and subjected to light microscopy using standard stains (IHC)on paraffin sections and reported according to modified Banff’s criteria.
▪︎Adequate biopsies with immunological injuries were categorized as:
Group 1: pure ABMR,
Group 2: combined ABMR with concurrent
T-cell-mediated rejection (TCR)
Group 3: pure TCR.
▪︎Groups 1 and 2 were further subgrouped as C4d positive (Group 1a and 2a) or C4d
negative (Group 1b and 2b).
▪︎ Graft function was measured by serum creatinine (SCr) level (mg/dL).
▪︎ 43.3% of biopsies revealed immunological injury. Of these, 27.7% of the biopsies revealed pure ABMR (Group 1), 60.6% revealed combined ABMR with
TCR (Group 2), and 11.3% revealed pure TCR (Group 3).
▪︎The overall incidence of ABMR (pureABMR + ABMR with TCR) was 36.27%, ofwhich C4d-negative rejections were 18.48% and 18.7% in Group 1 and Group 2, respectively.
▪︎The mean SCr at the end of three years follow-up in patients with C4d-negative rejections was comparatively higher.
☆Discussion:
______________
▪︎C4d staining is an important part of the diagnostic workup in suspected cases of ABMR.
▪︎ The most recent Banff meeting update highlights two major phenotypes of ABMR.
1) Appears early in the posttransplant period in a presensitized patient and is more likely to be C4d positive.
2) Develops late posttransplant and is due to de novo DSA development and is likely to be C4d negative (Class II HLA molecules may be responsible).
▪︎C4d-negative ABMR usually occurs >12 months after transplantation but can occur
acutely in highly sensitized patients with persistent DSAs (even after desensitization).
▪︎ Causes of C4d negativity:
1) Technical issues.
2) Different methods of C4d detection
3) Poor complement fixation by some DSAs
4) Complement-independent PW of ABMR
5) Autoantibodies to the angiotensin II type 1 receptor
6) AR due to Fc receptors on NK cells (FcRIIA)
▪︎C4dnegative ABMR tends to occur slightly later posttransplant and has a subclinical presentation with better graft outcome than C4d positive ABMR.
▪︎ C4d-negative ABMR is much less than C4d-positive ABMR (72.3%).
▪︎ C4d–negative ABMR when occurs alone, usually presents later than C4d-positive ABMR; however, if concurrent TCR is also seen then it usually presents earlier than
C4d-positive ABMR.
▪︎ The overall graft survival is better in C4d-negative ABMR group than the cohort of
C4d-positive ABMR
▪︎C4d-negative ABMR does have a poorer graft function as reflected by the higher
serum creatinine levels.
▪︎There is a subset of patients also needs to be treated aggressively like those with C4d-positive ABMR irrespective of concurrent or absence of TCR.
▪︎ There is a possibility of C4d negative converting to C4d positive
☆Conclusion:
_______________
▪︎The demographic profile or clinical symptoms are not helpful in determining the risk factors for the development of C4d-negative ABMR.
▪︎C4d-positive and C4d-negative ABMR shows similar frequencies of concurrent cell mediated rejection and both can occur early or late posttransplant.
▪︎ Graft outcomes were comparatively better in C4d-negative ABMR as compared
to C4d-positive ABMR
▪︎ Longitudinal prospective studies are required to know the clinical significance and long-term outcome of C4d-negative ABMR.
▪︎Molecular studies would be helpful to determine antibodies responsible for C4d-negative ABMR.
__________________
☆What is the kind of this study?
Retrospective cohort study
· Please summarise this article A retrospective study ,
· Jan 2013 to Dec 2015 , 987 transplanted patients underwent renal allograft biopsies, under LM and C4dstaining. S.creatinine was done at time of biopsy . Flow up rage from 5 days to 8 years. · Inclusion Criteria: Evidence of rejection in allograft biopsies. Exclusion Criteria: ABO-incompatible transplant and patient lost their follow up.
Adequate biopsies with immunological injuries were categorized
C4d positive AMR: Circulating DSA with biopsy showing glomerulitis, peritubular capillaritis and C4d>0 on IHC.
C4d negative AMR: Circulating DSA with biopsy showing glomerulitis, peritubular capillaritis and C4d 0 on IHC.
TCR : Biopsy with tubulitis (t>1), vasculitis (v>0) and interstitial inflammation (i≥2).
AMR with TCR: Combination of TCR and g≥1.
Outcomes: death censored graft loss (primary endpoint), patient death or dialysis dependency (secondary endpoint)
.Results:
· 43% of the biopsies revealed an immunological injury
27% of showed pure ABMR.
60% combined TCMR+ABMR.
11% pure TCMR.
18% of ABMR were C4d negative Limitations:
1. No biopsy flow up done for C4d negative patients
2. No DSA level flow up done
3. Conclusion:
The incidence of C4d negative ABMR is low compare to C4d positive ABMR
Graft outcomes were better in C4d negative ABMR patients. · · What is the kind of this study?
· Cohort retrospective study · What is the level of evidence?
· Level 3
This is a level III retrospective research to investigate the incidence and outcome of C4d-negative ABMR in renal allograft biopsies from 987 renal transplant patients.
A total of 987 renal allograft biopsies from 987 renal transplant recipients were analyzed.
Using light microscopy and C4d immunohistochemistry on paraffin sections, all renal allograft biopsy samples were investigated and reported using modified Banff’s criteria for renal allograft biopsy samples. The findings of the renal transplant biopsy were separated into groups. Group 1 contains just ABMR, Group 2 has both ABMR and concurrent T-cell-mediated rejection (TCR), and Group 3 contains only TCR. Groups 1 and 2 were further separated into C4d positive (Groups 1a and 2a) and C4d negative (Groups 1b and 2b) individuals (Groups 1b and 2b). The amount of serum creatinine (SCr) (milligrams per deciliter of blood) was used to assess graft function.
Although the demographic profile and clinical symptoms are important, they are not useful in identifying the risk factors for developing C4d-negative ABMR.
Pre-biopsy serum creatinine levels were as follows:
When the serum creatinine levels of each cohort were compared, it was shown that patients who tested positive for pure C4d had a higher SCr level (2.74 0.35) than patients in any other subgroup.
Estimation of donor-specific antibodies in a qualitative manner:
Patients with ABMR test positive 100 per cent of the time. DSA was not present in any of the cases involving TCR.
Match with human leukocyte antigen:
In all groups, the better the HLA match, the greater the transplant survival rate was seen.
The following are the current serum creatinine levels:
The SCr levels in patients with C4d– positive ABMR were lower than those in patients with C4d– negative ABMR because the C4d– positive group received more intensive plasmapheresis treatment than the C4d– negative group. This demonstrates that if C4d-negative damage is not treated quickly, it might have a deleterious impact on graft function and even cause graft attrition over the long term.
Graft survival is a matter of degree.
When comparing the C4d-positive and C4d-negative groups, it appears that graft loss is greater in the C4d-positive group (5.2 per cent vs. 2.5 per cent).
C4d-negative However, in highly sensitized individuals with chronic DSAs, ABMR may manifest themselves within 12 months of transplantation, rather than over time (even after desensitization).
The following are the causes of C4d negativity:
Technical concerns relating to the type of fixative employed led to the employment of several techniques of C4d detection.
Some DSAs have poor complement fixation, and the ABMR route is not dependent on complement fixation.
Autoantibodies to the angiotensin II type 1 receptor have also been linked to graft loss and fibrinoid necrosis, which are similar to alloantibody-mediated rejection except for the lack of PTC C4d, which is frequent in alloantibody-mediated rejection.
Results: The total incidence of ABMR (pure ABMR plus ABMR with TCR) was 36.27 per cent, with C4d-negative rejections accounting for 18.48 per cent and 18.7 per cent in Groups 1 and 2, respectively. Conclusions: Patients with C4d-negative rejections had a significantly higher mean SCr at the end of three years of follow-up than those with C4d-positive rejections. In Banff’13, C4d-negative ABMR was listed for the first time. It has a low incidence and generally manifests very early after transplantation, but it has a better result than C4d-positive ABMR.
-The recognition of the transplanted organ as foreign is mediated by complex
immunologic pathways, which can be divided into cellular (T-cell mediated) and humoral (antibody/B cell-mediated) pathways.
-biopsy is the gold standard and provides valuable insights into the pathogenesis of early and late allograft injury.
– Acute rejection (AR) episodes are a major determinant of renal allograft (RA )survival and the long-term outcome of renal transplant (RT).
– Acute antibody-mediated rejection (ABMR) occurs in 5%–7% of all RTs and is responsible for 20%–48% of AR episodes.
-The prevalence of chronic ABMR varies from 5% at one year to 20% at five
years.
-ABMR generally has a worse prognosis .
-The most common mechanism underlying ABMR is antibody response that originates from previous antigenic exposure or de novo development of DSA. Other implicated causes could be the presence of antibodies that are directed against HLA/major histocompatibility complex (MHC) class I and II antigens. Antibodies can be directed against other donor-specific antigens such as MHC Class I related chain A antigens, MHC Class I-related chain B antigens, platelet-specific antigens, molecules of the renin–angiotensin pathway, and polymorphisms involving chemokines and their receptors.
-C4d is a fragment of complement C4b, an activation product of the classic complementpathway. Detection of C4d is regarded as an indirect sign, a “footprint” of an antibody response.
-No functional role of C4d has been reported.
-C4d deposition is strongly associated with circulating antibody to donor HLA Class I/II antigens and is currently the best single marker of complement-fixing
circulating antibodies to the endothelium.
-It is a retrospective study performed from January 2013 to December 2015.
-A total of 987 renal allografts (RA) biopsies obtained from 987 RA recipients were studied.
-Graft function was measured by SCr levels (mg/dL).
– Formalin-fixed biopsy specimens were processed for light microscopy and C4d immunohistochemistry as per the manufacturer’s protocol.
-Biopsies negative for C4d were re-stained to rule out analytical errors associated with the staining procedure.
-Optimal biopsy was defined as a specimen with at least 10 nonsclerotic glomeruli and two arteries, a marginal biopsy having seven to nine glomeruli and one artery, and a minimally acceptable biopsy having seven glomeruli and one artery.
-Definition of C4d-positive ABMR – ABMR was defined if there was the presence of circulating DSA along with glomerulitis and peritubular capillaritis on histopathology with the presence of C4d deposition across the PTC membranes with an intensity of ≥2+ on immunofluorescence and C4d >0 by immunohistochemistry on paraffin-embedded sections.
-Definition of C4d-negative ABMR – C4dnegative ABMR was defined as biopsies with C4d <2 on immunofluorescence or C4d >0 on IHC, ptc> 0 and g >0, g+ptc ≥2, or g>0 or PTC >0 and acute TMA in the absence of any other cause of TMA.
-The most recent Banff highlights two major phenotypes of ABMR. The
first type appears early in the posttransplant period in a presensitized patient and is more likely to be C4d positive. The second type develops late posttransplant and is due to de novo DSA development and is likely to be C4d negative. The second phenotype is an important factor in late graft loss.
– C4d-negative ABMR usually occurs >12 months after transplantation but can occur acutely in highly sensitized patients with persistent DSAs (even after desensitization).
-Autoantibodies to the angiotensin II type 1 receptor have also been associated with graft loss and fibrinoid necrosis that resembles alloantibody-mediated
rejection except for the common absence of PTC C4d.
-Several tudies have concluded that C4d-negative ABMR tends to occur slightly later post transplant and has a subclinical presentation with better graft outcome than C4dpositive ABMR.
-The overall incidence of ABMR was higher in this study that could be attributed to various causes such as transplantation across HLA barriers, multiple transfusions, presensitized patients, and multiple pregnancies in women apart from low hygienic conditions.
-The overall incidence of C4d-negative ABMR in this study was 18.2%, which is much less than C4d-positive ABMR (72.3%). C4d–negative ABMR when occurs alone, usually presents later than C4d-positive ABMR (1.51 vs. 1.32 months) .
-The overall graft survival of patients with C4d-negative ABMR was 97.5% as againstC4d-positive ABMR which was 95.5%. Although associated with better graft survival, the C4d-negative ABMR does have a poorer graft function as reflected by the higher serum creatinine levels. What is the kind of this study?
Retrospective study What is the level of evidence?
3
Summary: Introduction:
Kidney transplant is the best renal replacement therapy for patients with end stage kidney disease associated with better Quality of life, improved graft survival and overall patient survival. kidney graft dysfunction can be triggered by many immunological and non-immulogical factors ,immunological triggers are common and complex including humoral and cellular immune response which can be lead to different types of allograft rejections ranging from hyper-acute rejection , accelerated rejection , acute and chronic active rejections , graft biopsy still considered as the best method for the diagnosis of the different types allograft dysfunction. Aim of the study:
·To measure the frequency of c4d negative ABMR and the overall graft function and graft survival as a final outcome. Method and materials:
·this study reviewing the histopathology of 987 renal allograft biopsies retrospectively including LM and C4D staining by using IHC from paraffin block, Biopsies negative for c4d were restrained to avoid technical error
· study period from Jan, 2013- dec,2016.
They divided the cases in 3 main groups, group one pure ABMR, group 2 mixed rejection (ABMR, ACR), Group 3pure ACR ,Group 2 according to c4d staining they further subdivided in two groups g1a,1b c4d+ve and c4d negative 2a,2b, 5 pathologist independently reviewed the graft biopsies , based on the modified Banff criteria updated in 2013 for the classification of graft injury , membranous and SLE slides used as positive controls , they include all specimens with confirmed allograft injury including acute TMA , 5.3% inadequate biopsies were excluded for interpretations, also they excluded those with ABOI transplantation , and those who lost for follow up.
Antirejection protocols documented according to the type of rejection
Pure ACR, treated with PMP 3-5 Days, ATG in resistant ACR 1.5MG /kg
Pure C4D +VE ABMR (PP, IVIG, plus Bortezomib 1.3mg/kg in 4 doses or single dose rituximab
Mixed rejection (PMP, ATG, PP)
C4D-ve ABMR treated with monotherapy either bortezomib or rituximab, no PP
Results:
43.3% biopsies showed immunological injury, 27.7% pure ABMR
Mixed rejection 60%, 11.3% pure ACR, overall ABMR (PURE, MIXED) was 36.27%
While c4 negative ABMR was lower than C4D+ve ABMR only 18.4and 18.6in group1,2
Majority of patients are young male adults with median age of 33
Mean post – transplant FU period in months was inconstant between the 3 groups with shortest in C4D+ve ABMR group1 , followed by C4D-ve ABMR ,TCR Group2b, and even after one year fu they found that c4d +ve ABMR is predominate type 75.5% compared to 20% of c4d-ve ABMR.Pure C4D positive ABMR more predominate and more sever acute graft dysfunction at time of presentation and up on last FU however C4 -VE ABMR was higher compared to the control group , DSA 100% positive in ABMR Graft survival outcome:
Graft survival better in C4D-ve despite higher mean SCR level in this group and received less aggressive therapy compared to C4D +ve ABMR Discussion:
The updated Banff classification highlighted two major phenotypes of ABMR
-phenotype 1 C4D +VE ABMR early post transplantation period in highly sensitized patients
– Phenotype 2 C4D -ve ABMR, late post-transplant, due to denovo anti HLA class11 antibodies and associated with late graft loss but again can occur acutely in highly sensitized patients with persistent DSA despite desensitization, complement independent direct endothelial damage by antibody dependent cell mediated cytotoxicity including NK, neutrophils. IN this cohort group the incidence of ABMR is higher than TCR with C4D -ve ABMR only 18% while C4D +VE predominates of total 72%, this can be explained by higher rate of presensitization with blood transfusion, pregnancies, nonadherence. The prevalence C4D negative ABMR reported similar rate or even higher in some studies as high as 49% (34,33).
Although C4D -ve ABMR shows better graft survival rate in this study but its overall associated with progressive graft loss over time.
Limitations:
Retrospective design
No biopsies for the C4D negative ABMR on FU
Quantitative DSA not available
Aim of the study:
· To measure the frequency of c4d negative ABMR and the overall graft function and survival as an outcome. Method and materials:
· this study reviewing the histopathology of 987 renal allograft biopsies retrospectively including LM and C4D staining by using IHC from paraffin block, Biopsies negative for c4d were restrained to avoid technical error
· study period from Jan, 2013- dec,2016.
They divided the cases in 3 main groups, group one pure ABMR, group 2 mixed rejection (ABMR, ACR), Group 3pure ACR ,Group 2 according to c4d staining they further subdivided in two groups g1a,1b c4d+ve and c4d negative 2a,2b, 5 pathologist independently reviewed the graft biopsies , based on the modified Banff criteria updated in 2013 for the classification of graft injury , membranous and SLE slides used as positive controls , they include all specimens with confirmed allograft injury including acute TMA , 5.3% inadequate biopsies were excluded for interpretations, also they excluded those with ABOI transplantation , and those who lost for follow up.
Antirejection protocols documented according to the type of rejection
Pure ACR, treated with PMP 3-5 Days, ATG in resistant ACR 1.5MG /kg
Pure C4D +VE ABMR (PP, IVIG, plus Bortezomib 1.3mg/kg in 4 doses or single dose rituximab
Mixed rejection (PMP, ATG, PP)
C4D-ve ABMR treated with monotherapy either bortezomib or rituximab, no PP
Results:
43.3% biopsies showed immunological injury, 27.7% pure ABMR
Mixed rejection 60%, 11.3% pure ACR, overall ABMR (PURE, MIXED) was 36.27%
While c4 negative ABMR was lower than C4D+ve ABMR only 18.4and 18.6in group1,2
Majority of patients are young male adults with median age of 33
Mean post – transplant FU period in months was inconstant between the 3 groups with shortest in C4D+ve ABMR group1 , followed by C4D-ve ABMR ,TCR Group2b, and even after one year fu they found that c4d +ve ABMR is predominate type 75.5% compared to 20% of c4d-ve ABMR
Pure C4D positive ABMR more predominate and more sever acute graft dysfunction at time of presentation and up on last FU however C4 -VE ABMR was higher compared to the control group ,
DSA 100% positive in ABMR
Graft survival outcome:
Graft survival better in C4D-ve despite higher mean SCR level in this group and received less aggressive therapy compared to C4D +ve ABMR Discussion:
The updated Banff classification highlighted two major phenotypes of ABMR
-phenotype 1 C4D +VE ABMR early post transplantation period in highly sensitized patients
– Phenotype 2 C4D -ve ABMR, late post-transplant, due to denovo anti HLA class11 antibodies and associated with late graft loss but again can occur acutely in highly sensitized patients with persistent DSA despite desensitization, complement independent direct endothelial damage by antibody dependent cell mediated cytotoxicity including NK, neutrophils.
In this cohort group the incidence of ABMR is higher than TCR with C4D -ve ABMR only 18% while C4D +VE predominates of total 72%, this can be explained by higher rate of presensitization with blood transfusion, pregnancies, nonadherence
The prevalence C4D negative ABMR reported similar rate or even higher in some studies as high as 49% (34,33).
Although C4D -ve ABMR shows better graft survival rate in this study but its overall associated with progressive graft loss over time. Limitations:
Retrospective design
No biopsies for the C4D negative ABMR on FU
Quantitative DSA not available
Type of the study
retrospective cohort study with FU outcome
level of evidence :
2B
Heba Wagdy
3 years ago
Appropriate management of renal allograft dysfunction is important for long-term graft survival, Biopsy is still the gold standard for diagnosis of the cause of early and late graft injury.
Acute antibody mediated rejection (AMR) is responsible for 20-48% of acute rejection episodes and has worse prognosis than acute cellular rejection (ACR)
AMR is caused by alloantibodies against donor specific HLA molecules or other cell antigens, they attack peritubular and glomerular capillaries.
C4d is a fragment of complement C4b, an activation product of the classic complement pathway, it binds covalently to endothelial cell surface and extracellular matrix component making a stable molecule easily detected by immunohistochemistry.
Detection of C4d is considered “a footprint” of an antibody interaction and is the best single marker of complement fixing circulating antibodies.
AMR presents with 2 major phenotypes:
first type appears early post transplant in pre-sensitized patients and more likely to be C4d positive
second type appears late post transplant due to de novo DSA and is likely to be C4d negative and may occur acutely in highly sensitized patients with persistent DSA.
C4d staining is a tool for diagnosis of antibody mediated graft injury either early or late post transplant.
C4d negative may be due to technical issues, poor complement fixation by some DSAs, complement independent pathway of AMR, Autoantibodies to angiotensin II type 1 receptor are associated with fibrinoid necrosis resembling AMR but with absent C4d deposition.
Studies showed that C4d negative AMR tend to occur late post transplant with subclinical presentation with better graft outcome than C4d positive AMR Materials and methods:
Retrospective study aimed to determine the incidence and clinical outcome of C4d negative AMR
It included indicated diagnostic renal allograft biopsies that revealed immunological rejection and biopsies from patients with ABO incompatible transplants.
Biopsies were processed for light microscopy and C4d immunohistochemistry and were classified according to Revised Banff’ 13 diagnostic categories. Study design:
The biopsies were categorized into 3 groups, group1 with biopsies revealing AMR, group2 with combined ACR and AMR and group 3 with ACR.
group1 and group2 were subdivided according to absence or presence of C4d deposition.
All patients with rejection received therapy according to type of rejection.
Patient death or dialysis dependency was the proposed endpoints of the study.
In this study, the incidence of C4d negative AMR was 18.2% while C4d positive was 72.3%
C4d positive and C4d negative AMR have similar frequency when concurrent cell mediated rejection is present and both may occur early or late post transplant.
C4d negative AMR was associated with better graft survival but with poorer graft function.
C4d negative AMR needs to be treated aggressively as C4d positive AMR irrespective of concurrence or absence of TCR
Prospective studies are needed to determine long term outcome of C4d negative AMR. Limitations:
C4d negative AMR wasn’t re-biopsied during follow up so couldn’t determine possibility of conversion of C4d negative to C4d positive.
Quantitative DSA estimation wasn’t done
It couldn’t determine risk factors for development of C4d negative type of study
Retrospective study level of evidence: 3
Batool Butt
3 years ago
Please summarise this article
Various immunological and non-immunological causes of renal allograft dysfunction (RAD) have been identified, of which humoral (antibody/B-cell mediated) and cellular (T-cell mediated)are the most common. Alloantibodies preferentially attack the peritubular and glomerular capillaries and T- cells infiltrate tubules and arterial endothelium-also both have different treatment options. ABMR have antibodies which can be due novo or preformed and directed against both HLA and non-HLA molecules.C4d deposition is associated with circulating antibody to donor HLA Class I/II antigens and is specific for complement fixing circulating antibodies to the endothelium.
A retrospective study conducted from January 2013 to December 2015,in which 934 patients with graft biopsies revealing immunological rejection were being included. In this study, biopsies were classified according to Revised Banff’13 diagnostic criteria’s. All the biopsies were compared for mean post transplant and mean follow-up time period in months. . S Creatinine levels (mg/DL) at the time of biopsy and at last follow-up, patient and graft survival were being compared. The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%, of which C4d-negative rejections were 18.48% and 18.7% in Group 1 (pure ABMR)and Group 2(combined ABMR with concurrent TCR), respectively. The mean Serum Cr at the end of three years follow-up in patients with C4d-negative rejections were comparatively higher. C4d-positive and C4d-negative ABMR shows similar frequencies of concurrent cell- mediated rejection and both can occur early or late post transplant. Graft outcomes were comparatively better in patients who developed C4d-negative ABMR as compared to C4d-positive positive ABMR.
Two major phenotypes of ABMR highlighted at the meeting :
1- Appears early in the post transplant period in a pre sensitized patient and is more likely to be C4d positive.
2- Develops late post transplant, is due to de novo DSA development, and is likely to be C4d negative. This type is most important, as it is responsible for late graft los
C4d negative result may be due to technical errors , different method of C4d detection ,poor complement fixation by DSA , complement independent pathway ABMR , antibodies against And II type 1 receptors , NK Fc receptors may have a role in acute rejection with C4d negative staining. Limitations of the study ;
1-No re-biopsy during follow-up.
2- No quantitative DSA estimation was done.
Renal allograft dysfunction is
common and critical for long term graft function and survival. So we need accurate diagnosis for appropriate management .
Various causes of immunological and nonimmunological of renal graft dysfunction have been identified.
And there are several types of rejection that can occur in a RA including hyperacute, accelerated acute, acute, and chronic active rejections.
The immune system has the capability to recognise the transplanted graft as foreign and mediate an immune response through complex immunological pathways, its divided into cellular and humoral pathways.
For diagnosis biopsy remains the gold standard and provides valuable insights
into the pathogenesis of early and late allograft injury.
Acute ABMR is seen in 5-7% of transplant recipients and makes up for 20-48% of all acute rejections. Antibodies in AMR act on peritubular and glomerular capillaries, while the T cells act on tubules and arterial endothelium in T cell mediated rejection.
Retrospective study was been conducted in 987 subjects to determine the incidence and outcome of C4d-negative ABMR. Biopsies were categorized through light microscopy and C4d immunohistochemistry into Group 1: pure ABMR, Group 2: combined ABMR with concurrent T-cell-mediated rejection (TCR), and Group 3: pure TCR. Groups 1 and 2 were further subgrouped as C4d positive (Group 1a and 2a) or C4d negative (Group 1b and 2b).
• Immunological injury was confirmed in 43.3% of these biopsies
• the most common form rejection was mixed rejection (60.6%), followed by pure ABMR (27.7%) and lastly pure TCMR (11.3%).
• C4d positive ABMR is more common (around 72%) thanC4d negative ABMR (around 18%)
• Patients with ABMR who are C4d positive usually present earlier(C4d negative ABMR tends to occur later
• Patients with ABMR who are C4d positive has lower graft survival (77.7%). than those who are C4d negative (90.2%), this means that it is a marker for severity and has a prognostic impact.
• C4d negative patients have a higher rate of developing transplant glomerulopathy if untreated
• Serum creatinine is found to be higher in C4d negative ABMR when compared to positive ones and this may be explained by the non-aggressive treatment of C4d negative ABMR
• This shows that C4d-negative injury does affect the graft function if not treated aggressively and causes graft attrition in the long-term .
All the patients who developed rejection
were subjected to antirejection regimen which was decided according to the type of rejection reported on biopsies. Patients who developed cellular rejection were treated with intravenous methyl prednisolone 500 mg/day for
3–5 days; rabbit anti-thymoglobulin (r-ATG) 1.5 mg/kg BW was added for resistant cases.
C4d-positive ABMR was treated with three sessions of plasmapheresis (with 40 mL/kg BW/session) followed by i.v. Ig 5 g on alternate days and subsequent bortezomib, 1.3 mg/m2 on days 1, 4, 7. and 11 or rituximab, 50–300 mg single dose.
For combined TCR + ABMR, methylprednisolone and rATG (in
doses mentioned above) were administered followed by plasmapheresis.
Patients with C4d-negative rejection were offered monotherapy with either bortezomib or rituximab. None of these patients underwent therapeutic plasma exchange.
The overall incidence of ABMR was higher this could be attributed to various causes such as transplantation across HLA barriers, multiple transfusions, presensitized patients, and multiple pregnancies in women apart from low hygienic conditions prevailing
no difference in demographic data of patients.
What is the kind of this study?
a retrospective cohort study
What is the level of evidence?
A retrospective study to determine the incidence and outcome of C4d- negative ABMR.
introduction,
Acute antibody mediated rejection (ABMR) generally has a worse prognosis and requires a different form of therapy than T-cell-mediated rejection (TCR).
Alloantibodies are now appreciated as important mediators of acute and chronic rejection, differing in pathogenesis or “nature,” from TCR.
Alloantibodies preferentially attack a different “location,” namely the peritubular and glomerular capillaries in contrast to T- cells which characteristically infiltrate tubules and arterial endothelium.
ABMR is caused by antibodies directed against donor-specific human leukocyte antigen (HLA) molecules or other cell antigens.
Study design,
A total of 987 renal allograft biopsies were studied using LM & C4d IHC on paraffin sections & reported according to modified Banff’s criteria.
Patients who underwent ABO-i transplants & those lost for FU were excluded (5.3%).
Of the remaining 934 allograft biopsies, 43.3% (404) revealed immunological injury on histology & were categorized as,
a. Group 1: pure ABMR (27.7%) b. Group 2: combined ABMR with concurrent TCMR (60.6%) c. Group pure TCR (11.3%). Groups 1 & 2 further divided into:
C4d positive (Group 1a & 2a)
C4d negative (Group 1b & 2b)
Anti rejection regimen used,
ACR: IV methylprednisolone 500 mg/day for 3–5 days; r-ATG 1.5 mg/kg BW added for resistant cases.
C4d-positive ABMR: 3 sessions of PP 40 mL/kg BW/ session followed by IV Igg 5 g DAD & subsequent bortezomib, 1.3 mg/m2 on days 1, 4, 7. & 11 or rituximab, 50–300 mg single dose.
Combined TCR + ABMR: methylprednisolone & rATG were given followed by PP.
C4d-negative rejection: only monotherapy (either bortezomib or rituximab).
Results
The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%, of which C4d-negative rejections were 18.48% and 18.7% in Group 1 and Group 2, respectively.
The mean SCr at the end of three years follow-up in patients with C4d-negative rejections was comparatively higher.
C4d-negative ABMR, recently included in Banff’13, has a low incidence, usually presents early after transplantation but carries better outcome than C4d-positive ABMR.
What is the kind of this study?
Retrospective study
What is the level of evidence?
level of evidence 3
20-30% of all acute rejection episodes involve a humoral component.
C4d is an indirect sign or footprint of antibody response in kidney allografts. however, antibody mediated rejection (AMR) still can occur with -ve C4d in renal biopsies.
C4d -ve AMR usually occur more than 12 months after renal transplantation and can occur acutely in highly sensitized patients with persistant DSAs.
C4d -ve AMR has subclinical presentation with better graft outcome than C4d +ve AMR
Causes of C4d-ve AMR include the following :
1- technical issues related to the type of the fixative of complement.
2- different methods of C4d detection.
3- poor complement fixation by some DSA.
4- complement-independent pathways of AMR.
5- auto-antibodies to Angiotensin II type 1 receptor. they can mediate graft loss and fibrinoid necrosis similar to rejection but with -ve C4d.
6- Fc receptors on NK cells may play a role in acute rejection with -ve C4d.
7- Loupy et al., reported significant proportion of patients with AMR transit between -ve C4d staining and +ve C4d deposition.
This retrospective study was done for evaluation of the incidence and outcome of C4d -ve AMR. it included 987 patients. the conclusion of this study was the following :
1- incidence of C4d -ve AMR is strickingly low. it is of 18.2 % in patients with AMR
2- clinical symptoms are not helpful in determining risk factors for C4d -ve AMR.
3- graft outcome was better in those with C4d -ve AMR. however, longitudinal studies are required to know the significance and long-term outcome.
4- molecular studies are helpful to determine antibodies responsible for C4d-ve AMR to design targeted therapies.
C4d negative Antibody mediated rejection: Pathologist’s Perspective and clinical outcome
This is retrospective study if indicated diagnostic renal allograft biopsies performed from July 2013 to December 2015. Patient whose biopsies demonstrated immunological rejection were induced but those who were ABOI and lost follow ups are excluded.
Study design
G1- pure ABMR , G1a- C4d positive, G1b- C4d negative
G2- combined TCR and ABMR, G2a- C4d positive and G2b- C4d negative
G3- pure TCMR
Outcome:
primary end point -Death censored graft loss
Secondary end point – patient death or dialysis dependency
Results
404 patients included and 80.4% Males.
Mean posttransplant period
Time: 5 days to 8 years
C4d + ABMR presented at 1.32 SD 0.28 months,
C4d -ABMR -TCR presented at 1.33 SD 0.15 months
C4d-ABMR- 1.51 SD 0.27 months
TCR pure- 1.61 SD 0.66 months
C4d -ABMR was 20.2% within one year
75.5% C4d + ABMR
When C4d- alone AR apperas later than C4d + ABMR but when it combined with TCR it present earlier 1.32 vs 1.51 months
C4d + ABMR when combined with TCMR – appears late than 1.98 months
Prebiopsy SCr
Highest in C4d + ABMR (2.5 SD0.24)
C4d-ABMR- 2.11 SD 0.32, but P value not significant
Subgroup noted pure C4d + ABMR had higher Scr level 2.74 SD 0.35
HLA mismatch
Better matched Ts had better graft survival
Current Scr
Mean Scr in C4d – was 1.85 SD 0.56 while Cd4+ was 1.93 SD 0.21
Mean SCr pure ABMR with C4d – was highest
Graft survival
Overal graft survival in C4d- was better
6.4% in C4d + ABMR had graft loss
TCR + C4d+ had 4.6% Graft loss
C4d-ABMR had 2.5%
C4d+ vs C4d- 5.2% vs 2.5%
In conclusion, C4d negative ABMR and C4d positive had similar frequencies of concurrent TCMR which can occur early or late.Graft outcome were comparatively better in C4d – cohort.
it is a retrospective study with level of evidences 3
Renal allograft dysfunction is a common and complex problem of Renal Tranx, several types of rejection that can occur in allograft including hyperacute, accelerated acute, acute, and chronic active rejections.
In spite of significant developments in the diagnostic methodologies for
diagnosis of allograft dysfuncation, biopsy still remains the gold standard and provides valuable insights into the pathogenesis of early and late allograft injury.
Alloantibodies preferentially attack a different “location,” namely the peritubular and glomerular capillaries in contrast to T- cells which characteristically infiltrate tubules and arterial endothelium. ABMR is caused by antibodies directed against donor-specific human leukocyte antigen (HLA) molecules or other cell antigens. The most common
mechanism underlying ABMR is an antibody response that originates from previous antigenic exposure or de novo development of DSA.
Feucht et al showed that peritubular capillary (PTC) C4d deposition in RT biopsies is strongly associated with a poor prognosis and raises the
possibility that antibodies are responsible for graft loss.
C4d is a fragment of complement C4b, an activation product of the classic complement pathway.
antibodies can lead to graft accommodation through upregulation of complement regulatory or cytoprotec-tive proteins. It depends on the specificity and concentration of the antibodies: high-titer DSAs may cause ABMR, while recipients with low-titer DSAs can develop accommo-
dation. Endothelial cells are targets for immunemediated assaults through antiendothelial cell antibodies. Study Place pathology department, Civil hospital campus, India from Jan 2013 to end of 2015. Type : a retrospective study 987 patients who underwent renal allograft biopsies, all samples are subjected to LM and C4d IHC. S.cr at time of biopsy and follow up is obtained. Time post-transplantation ranges from 5 days to 8 years. Inclusion Criteria: patients whose biopsy showed evidence of rejection. Exclusion Criteria: ABO-incompatible transplant, lost to follow up. Results:
43% of the biopsies revealed an immunological injury, 27% of them showed pure ABMR, 60% combined TCMR+ABMR,11% pure TCMR, 18% of ABMR were C4d negative ,Mean Scr at the C4d negative ABMR was higher as compared to C4d positive after 3 years of follow up, Overall graft survival was better in C4d negative patients.
conclusion:
The incidence of C4d negative ABMR is low in this study.
Graft outcomes were better in C4d negative ABMR patients.
It is aretrospective study level 3.
I. C4d-Negative Antibody-Mediated Rejection: A Pathologist’s Perspective and Clinical Outcome Please summarise this article This study retrospectively looked into the incidence & outcome of C4d-negative ABMR. A total of 987 renal allograft biopsies were studied using LM & C4d IHC on paraffin sections & reported according to modified Banff’s criteria. Patients who underwent ABO-i transplants & those lost for FU were excluded (5.3%). Of the remaining 934 allograft biopsies, 43.3% (404) revealed immunological injury on histology & were categorized as: 1. Group 1: pure ABMR (27.7%) 2. Group 2: combined ABMR with concurrent TCMR (60.6%) 3. Group pure TCR (11.3%). Groups 1 & 2 further divided into:
– C4d positive (Group 1a & 2a)
– C4d negative (Group 1b & 2b). Overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27% (C4d-negative rejections were 18.48% in Group 1 & 18.7% in Group 2). The incidence of C4d-negative ABMR was less than that of C4d-positive ABMR. Mean S.Cr at the end of 3 years FU in patients with C4d-ve rejections was comparatively higher. Anti-rejection regimen was given to all who developed rejection:
– ACR: IV methylprednisolone 500 mg/day for 3–5 days; r-ATG 1.5 mg/kg BW added for resistant cases.
– C4d-positive ABMR: 3 sessions of PP 40 mL/kg BW/ session followed by IV Ig 5 g DAD & subsequent bortezomib, 1.3 mg/m2 on days 1, 4, 7. & 11 or rituximab, 50–300 mg single dose.
– Combined TCR + ABMR: methylprednisolone & rATG were given followed by PP.
– C4d-negative rejection: only monotherapy (either bortezomib or rituximab). Outcome The mean S. Cr level in C4d-ve ABMR was higher compared to other groups. C4d+ve group was treated aggressively with PP compared to the C4d-ve group, so the SCr levels were less than in patients with C4d-ve ABMR. This shows that C4d-ve injury does affect the graft function if not treated aggressively & causes long-term graft loss. Graft survival was better in the C4d-ve group despite the higher mean S. Cr levels in this group (Fig 1 & 2). The incidence of C4d-ve ABMR was low & the demographic or clinical parameters were unhelpful in determining risk factors of development of C4d-ve ABMR. C4d+ve & C4d-ve ABMR show similar frequencies of concurrent CMR & both can occur early or late post-transplant. Although graft outcomes were better in patients with C4d-ve ABMR as compared to C4d+ve ABMR, however longitudinal prospective studies are needed to determine the clinical significance & long-term outcome of C4d-ve ABMR. Molecular studies might helpful to determine the responsible antibodies to guide treatment approach in this group & thus improve graft survival these patients. Definitions:
– C4d-positive ABMR: presence DSA+ glomerulitis & PTCs on histopathology + PTC C4d deposition with an intensity of ≥2+ on IF & C4d >0 by IHC.
– Acute TMA) in the absence of any other cause was also reported as ABMR.
– Glomerulitis (g>0) – defined as the % of glomeruli with leukocytes infiltration in the capillaries(Score g1, g2, & g3 if <25%, 26%–50%, & >50% of glomeruli affected).
– Peritubular capillaritis – % of PTCs in the cortex that contained neutrophils or mononuclear cells (ptc1: <10% PTCs revealed cells, ptc2:10% of PTCs in-filtrated by <5 cells, ptc3:10% of PTCs revealed >10 cells/PTC).
– C4d deposition scoring by IHC– positive if grade was >C4d0 (C4d1, C4d2, & C4d3 if 1–9%, 10%–50%, & >50% PTCs affected, respectively).
– C4d-negative ABMR – biopsies with C4d <2 on IF or C4d < 0 on IHC, ptc> 0 & g >0, g+ptc ≥2, or g>0 or ptc >0 & acute TMA in the absence of other causes.
– TCR – biopsies with tubulitis (t>1) along with vasculitis (v>0) &/or interstitial inflammation (i>2).
– ABMR with TCR – Biopsies with C4d-ve ABMR+ histological evidence of TCR also had g ≥1 to qualify the criteria of ABMR.
– DSA (anti– HLA Class I & II antibody specificity) performed using SABs on Luminex. MFI >1000 was considered positive.
file:///C:/Users/TOSHIBA/AppData/Local/Temp/msohtmlclip1/01/clip_image002.gif Figure 1. Kaplan–Meier survival plot depicting the renal allograft survival.
file:///C:/Users/TOSHIBA/AppData/Local/Temp/msohtmlclip1/01/clip_image004.gif Figure 2. Kaplan–Meier survival plot depicting the patient survival. What is the kind of this study? Hospital-based retrospective cohort study What is the level of evidence? Level II
1)summary:
Renal transplantation is optimal treatment option for ESRD patients.
graft loss is a challenge facing renal transplantation which it could be multi-factorial such as immunological causes include:acute graft rejection(ABMR&TCR).
Renal allograft biopsy is the gold standard tool for diagnosis of RAD.
In all renal TX the ABMR occur in 5 to 7% and 20 -48% of AR episodes.
ABMR has worse prognosis and its modalities of therapy is different from TCR,and is caused by antibody direct against DSA donor.
C4d is a fragment of complement of c4b activation product of the classical complement pathway.
Feucht et al suggested that capillary c4 deposition was an evident for humeral alloreactivity against the graft.
capillary c4d staning is pathogonamic for antiHLA alloantibody and this antibody can be mediated through complement dependent and independent mechanism.
This study performed in Hospital Campus and Ahmedabad, Gujarat, India to determine the incidence of c4d negative ABMR and evaluate their outcome in term of graft function and survival.
This retrospective study was carried on from 2013 to Dec 2015 for 934 graft biopsy and the graft function was monitored by Scr level(mg/dl) the technique was used c4d IHC formalin-fixed biopsy specement to examine by light microscopy.
The study include all patients revealed immunological rejection in graft biopsy but exclude:
ABO incompatible TX
patient whom lost follow up
Baniff13 diagnostic classify for RA biopsy is used in this study
Optimal adequate biopsy at least 10 glomeruli +2 arteries
marginal biopsy 7-9 glomeruli +1 artery
minimal acceptable biopsy 7 glomeruli +1 artery
presence of DSA ,c4d +ve,glomerulitis and capillaritis all are used to diagnosed ABMR
They used IHC parrafin in embedded to detect c4d deposition in PTC graded based on intensity c4d +ve if more than 0 and IF 2 or more
Score of glomerulitis based on%infiltration of leukocytes
based on type of rejection ,all patients received specific anti rejection therapy and c4d negative patients received either bortezomib or rituximab
The end point of patients in the study is death or patients dialysis dependent
Scr in c4d negative group was higher than c4d +ve group since c4d +ve group received aggressive anti rejection therapy
Factors influence the c4d negative result:
technical factors
poor complement fixing by DSA
complement independent pathway
auto antibody to the Angio 2 type 1 receptor
FC receptor and natural killer cells(Fc R11A) cause acute rejection
Denovo DSA when developed in late post transplant
Conclusion:
Incidence of c4d negative antibody mediated rejection
TCR has the same incidence of occurrence with c4d +ve and negative
Graft outcome better on c4d -ve than +ve
Requirement of prospective studies to know the clinical significance and long term outcome of c4d -ve ABMR
2) The type of study is retrospective study
3) level 3
Settings: pathology department, Civil hospital campus, India from Jan 2013 to Dec 2015.
Design: a retrospective study
Patients: 987 patients who underwent renal allograft biopsies, all samples are subjected to LM and C4d IHC. S.cr at time of biopsy and follow up is obtained. Time post-transplantation ranges from 5 days to 8 years.
Inclusion Criteria: patients whose biopsy showed evidence of rejection.
Exclusion Criteria: ABO-incompatible transplant, lost to follow up.
Ethical approval: Institutional review board.
Protocol: five pathologists independently reviewed the allograft biopsies done in the study period; all of the biopsies were subjected to LM examination & C4d IHC staining, biopsies negative for C4d were re-stained to rule out errors. Membranous/ lupus nephropathy slides were used as positive controls. Endothelial cells lining the medium calibre blood vessels were taken as an internal control. All biopsies were compared for mean post-transplant and mean follow up the time period in months. Scr levels at the time of biopsy and follow up were compared. The patients were divided into three groups: group 1 ABMR, group 2 combined TCMR & ABMR, and group 3 TCMR. Group 1 & 2 are subdivided into: C4d positive & negative. Treatment of rejection was decided according to the type of rejection.
Outcomes: death censored graft loss (primary endpoint), patient death or dialysis dependency (secondary endpoint).
Results:
43% of the biopsies revealed an immunological injury
27% of them showed pure ABMR.
60% combined TCMR+ABMR.
11% pure TCMR.
18% of ABMR were C4d negative
Mean Scr at the C4d negative ABMR was higher as compared to C4d positive after 3 years of follow up.
Overall graft survival was better in C4d negative patients.
Limitations:
The patients are not rebiopsed during follow up period to exclude the conversion from C4d negative to C4d positive rejection.
No DSA estimation was done
Conclusion:
The incidence of C4d negative ABMR is low in this study.
Graft outcomes were better in C4d negative ABMR patients.
This is the retrospective study of level III to determine the incidence and outcome of C4d-negative ABMR where the 987 renal allograft biopsies from 987 renal transplant recipients were studied.
All renal allograft biopsy samples were examined by light microscopy using standard stains and C4d immunohistochemistry on paraffin sections and reported according to modified Banff’s criteria. The renal graft biopsy results divided into groups.Group 1: pure ABMR, Group 2: combined ABMR with concurrent T-cell-mediated rejection (TCR), and Group 3: pure TCR. Groups 1 and 2 were further subdivided as C4d positive (Group 1a and 2a) or C4d negative (Group 1b and 2b). Graft function was measured by serum creatinine (SCr) level (mg/dL).
Inclusion criteria: Patients with biopsies revealed immunological rejection were included in the study. Exclusion criteria: Patients who underwent ABO incompatible transplants and those who were lost for follow- up were excluded from the study.
-Adequacy of graft biopsy specimen defined as with at least 10 nonsclerotic glomeruli and two arteries. Specimens with less than 7 glomeruli or no artery considered inadequate biopsy for interpretation.
Definition of C4d-positive ABMR :was defined if there was the presence of circulating DSA along with glomerulitis and peritubular capillaritis on histopathology with the presence of diffuse C4d deposition across the PTC membranes with an intensity of ≥2+ on immunofluorescence and C4d >0 by immuno- histochemistry on paraffin sections.
Results:
The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%, of which C4d-negative rejections were 18.48% and 18.7% in Group 1 and Group 2, respectively. The mean SCr at the end of three years follow-up in patients with C4d-negative rejections was comparatively higher. C4d-negative ABMR, recently included in Banff’13, has a low incidence, usually presents early after transplantation but carries better outcome than C4d-positive ABMR.
The most recent Banff meeting update high- lights two major phenotypes of ABMR. The first type appears early in the posttransplant period in a presensitized patient and is more likely to be C4d positive.The second type develops late posttransplant and is due to de novo DSA development and is likely to be C4d negative.
C4d is a fragment of complement C4b, an
activation product of the classic complement pathway.
Detection of C4d is
regarded as an indirect sign, a “footprint” of an antibody response.
C4d deposition is strongly associated with circulating antibody to donor HLA Class I/II antigens and is currently the best single marker of complement-fixing circulating antibodies to the endothelium.
Antibodies can mediate
endothelial injury through complement-depen-
dent and independent mechanisms.
On the other hand, under certain conditions, antibodies can lead to graft accommodation through upregulation of complement regulatory or cytoprotective proteins.
It depends on the specificity and
concentration of the antibodies: high-titer
DSAs may cause ABMR, while recipients
with low-titer DSAs can develop accommodation.
A total of 987 renal allograft (RA) biopsies obtained from 987 RA recipients were studied from January 2013 to January 2016. All samples were subjected to light microscopy using standard stains and C4d immunohistochemistry on paraffin sections and reported according to modified Banff’s criteria. Adequate biopsies with immunological injuries were categorized as Group 1: pure ABMR, Group 2: combined ABMR with concurrent T-cell-mediated rejection (TCR), and Group 3: pure TCR. Groups 1 and 2 were further subgrouped as C4d positive (Group 1a and 2a) or C4d negative (Group 1b and 2b). Graft function was measured by serum creatinine (SCr) level (mg/dL). Of the 987 biopsies, 43.3% (404) biopsies revealed immunological injury. Of these, 27.7% of the biopsies revealed pure ABMR (Group 1), 60.6% revealed combined ABMR with TCR (Group 2), and 11.3% revealed pure TCR (Group 3). The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%, of which C4d-negative rejections were 18.48% and 18.7% in Group 1 and Group 2, respectively. The mean SCr at the end of three years follow-up in patients with C4d-negative rejections was comparatively higher. C4d-negative ABMR, recently included in Banff’13, has a low incidence, usually presents early after transplantation but carries better outcome than C4d-positive ABMR.
The demographic profile or clinical symptoms are not helpful in determining the risk factors for the development of C4d-negative ABMR
Prebiopsy serum creatinine levels:
On comparing the serum creatinine levels of each subgroup, it was observed that patients with pure C4d positive had a higher SCr level (2.74 ± 0.35) than any other subgroup.
Qualitative donor-specific antibodies estimation:
100% positivity in patients having ABMR. DSA was absent in all the cases with TCR.
Human leukocyte antigen match:
the better the HLA match, better was the graft survival in all the groups.
Present serum creatinine levels:
Since the C4d– positive group was treated aggressively with plasmapheresis as compared to the C4d- negative group, the SCr levels tended to be less than in patients with C4d-negative ABMR. This shows that C4d-negative injury does affect the graft function if not treated aggressively and causes graft attrition in the long-term .
Graft survival:
graft loss appears to be higher in the C4d-positive group as compared to the C4d-negative group (5.2% vs. 2.5%).
C4d-negative ABMR usually occurs
>12 months after transplantation but can occur
acutely in highly sensitized patients with
persistent DSAs (even after desensitization).
causes of C4d negativity:
Technical issues related to type of fixative
used
different methods of C4d detection.
poor complement fixation by some DSAs, complement-independent pathway of ABMR
Autoantibodies
to the angiotensin II type 1 receptor have also
been associated with graft loss and fibrinoid
necrosis that resembles alloantibody-mediated
rejection except for the common absence of
PTC C4d.
Fc receptors on NK cells (FcRIIA)
may also play a role in AR, and it is possible
that some examples of AHR in biopsies that
lack C4d are due to this mechanism.
The overall incidence of C4d-negative ABMR in this study was 18.2%, which is much less than C4d-positive ABMR (72.3%). C4d–negative ABMR when occurs alone, usually presents later than C4d-positive ABMR (1.51 vs. 1.32 months); however, if concurrent TCR is also seen then it usually presents earlier than C4d-positive ABMR (1.33 vs. 1.98 months).
This was an Institutional Review Board approved retrospective study.
C4 d negative antibody mediated rejection a pathologist perspective and clinical outcome
Please summarise this article
Renal graft dysfunction is major concern post kidney transplant and it can be categorised into 4 forms. This can be hyper acute , accelerated acute or chronic rejection. Rejection can be categorised into either cell mediated rejection or antibody mediated rejection and any form of rejection has poor outcome. In general the acute antibody mediated rejection is seen in about 5 to 7% of kidney transplant recipients and makes up about 20 to 48% of all acute rejections. In case of antibody mediated rejection antibodies act on Peri tubular capillaries while in case of cell mediated rejection it occurs on due to effect of T cells on endothelium
C 4d is a product of classical compliment cascade and it attaches to endothelial cell surface C4 d detection is a sign of antibody mediated rejection while there can be a group of patients who have AMR are with negative CD4
Material and methods: This was a retrospective study between January 2013 to December 2015 and the purpose of study was to define the incidents an outcome of C4d negative rejection. In this study a total of 987 renal allograft biopsies were done. The samples were processed under light microscope with standard stains and immunohistochemistry on paraffin sections and reported it according to modified Banffs criteria . Group One -was pure antibody mediated rejection Group 2- was combined antibody mediated rejection with concurrent TCR Group 3 -pure T cell mediated rejection. Group One and two were further subdivided into C4d positive and C4d negative .
Definition of Cd4 positive and C4d negative ABMR-
C4d positive- Positive DSA with glomerulitis and peritubular capilliritis. Positive C4d across PTC membrane . Thrombotic microangipathy with no other cause was labelled as positive for ABMR.
C4d negative- Negative C4d on IHC and acute TMA with no other cause
Results
60.0% had mixed rejection, Pure ABMR in 27.7 and 11.3 had TCMR. Incidence of TCMR was similar in both C4d positive and C4d negative cases. In ABMR, C4d positivity was seen in 82% and negativity in 28%. C4d positive ABMR had earlier presentation than C4d negative . Serum creatinine was better in C4d positive group like due to better treatment in this group. The study confirmed the prognostic value of C4d positivity . Graft survival was 77.7% in C4d positive group while it was 90.2 in negative group. Incidence of TG was high in C4d negative group.
C4d-Negative Antibody-Mediated Rejection: A Pathologist’s Perspective and Clinical Outcome
Various causes of immunological and nonimmuno logical renal allograft dysfunction (RAD) have been identified. There are several types of rejection that can occur in a RA including hyperacute, accele rated acute, acute, and chronic active rejections. The recognition of the transplanted organ as foreign is mediated by complex immunologic pathways, which in general terms can be divided into cellular (T-cell me- diated) and humoral (antibody/B-cell mediated) pathways.
Acute antibody–mediated rejection (ABMR) generally has a worse prog- nosis and requires a different form of therapy than T-cell-mediated rejection (TCR). Alloantibodies are now appreciated as important mediators of acute and chronic rejection, differing in pathogenesis or “nature,” from TCR. Alloantibodies preferentially attack a different “location,” namely the peritubular and glomerular capillaries in contrast to T- cells which characteristically infiltrate tubules and arterial endothelium. ABMR is caused by antibodies directed against donor-specific human leukocyte antigen (HLA) molecules or other cell antigens.
Showed that peritubular capillary (PTC) C4d deposition in RT biopsies is strongly associated with a poor prognosis and raises the possibility that antibodies are responsible for graft loss. C4d deposition is strongly associated with circulating antibody to donor HLA Class I/II antigens and is currently the best single marker of complemente fixing circulating antibodies to the endothelium. Capillary C4d deposition was an evidence for humoral alloreactivity against the graft. Demonstrated that staining of allograft biopsies for the fragment C4d is a specific and reliable method for identifying lesions due to allo-antibodies against HLA Class I and/or Class II antigens. The specificity of capillary C4d staining for anti-HLA alloantibody-depen- dent graft injury was further demonstrated recently by Böhmig. ABMR presents most of the time as severe allograft dysfunction and its prompt diagnosis and optimal treatment are essential.
This was an Institutional Review Board approved retrospective study of indicated diag- nostic RABs performed from January 2013 to December 2015. The demographic data regarding patient age, gender, indication for RA biopsy, and serum creatinine were collected. Graft function was measured by SCr levels (mg/dL). Formalin-fixed biopsy specimens were pro- cessed for light microscopy and C4d immuno- histochemistry as per manufacturer’s protocol. Membranous/lupus nephropathy slides were used as positive controls. Endothelial cells lining the medium caliber blood vessels were taken as internal control. Patients whose biopsies revealed immunological rejection were included in the study. Patients who underwent ABO incompatible transplants and those who were lost for follow- up were excluded from the study.
Histological categories were classified as per Revised Banff’13 diagnostic categories for RA biopsies. Definition of C4d-positive ABMR – ABMR was defined if there was the presence of circulating DSA along with glomerulitis and peritubular capillaritis on histopathology with the presence of C4d deposition across the PTC membranes. Others categories were defined based on the characteristics already mentioned: glomerulitis, peritubular capillaritis, C4d position. All the biopsies were compared for mean posttransplant and mean follow-up time period in months. SCr levels (mg/dL) at the time of biopsy and at last follow-up were compared. After analyzing the light microscopic features, the biopsies were categorized.
All the patients who developed rejection were subjected to antirejection regimen which was decided according to the type of rejection reported on biopsies. Patients who developed cellular rejection were treated with intra- venous methyl prednisolone 500 mg/day for 3–5 days; rabbit anti-thymoglobulin (r-ATG) 1.5 mg/kg BW was added for resistant cases. C4d-positive ABMR was treated with three sessions of plasmapheresis (with 40 mL/kg BW/session) followed by i.v. Ig 5 g on alter- nate days and subsequent bortezomib, 1.3 mg/m2 on days 1, 4, 7, and 11 or rituximab, 50–300 mg single dose. For combined TCR + ABMR, methylprednisolone and rATG (in doses mentioned above) were administered followed by plasmapheresis. Patients with C4d-negative rejection were offered monotherapy with either bortezomib or rituximab. None of these patients underwent therapeutic plasma exchange.
The most recent Banff meeting update high lights two major phenotypes of ABMR. The first type appears early in the posttransplant period in a presensitized patient and is more likely to be C4d positive. The second type develops late posttransplant and is due to de novo DSA development and is likely to be C4d negative. The second phenotype is an important factor in late graft loss; however, not many studies have been published regarding the clinical significance of this subset of patients. It appears that Class II HLA molecules may be responsible and that much of the endothelial damage is mediated by NK cells and, to a lesser extent, monocytes and neutro- phils; antibody-dependent cell-mediated cyto- oxicity. C4d-negative ABMR usually occurs >12 months after transplantation but can occur acutely in highly sensitized patients with persistent DSAs (even after desensitization).
The incidence of C4d-negative ABMR is strikingly low in our study. The demographic profile or clinical symptoms are not helpful in determining the risk factors for the development of C4d-negative ABMR. In our study, C4d-positive and C4d-negative ABMR shows similar frequencies of concurrent cell- mediated rejection and both can occur early or late posttransplant. Graft outcomes were com- paratively better in the cohort of patients who developed C4d-negative ABMR as compared to those who have C4d-positive ABMR. However, longitudinal prospective studies are required to know the clinical significance and long-term outcome of C4d-negative
What is the kind of this study?
This article is a primary study , in retrospective cohort.
C4d-Negative Antibody-Mediated Rejection: A Pathologist’s Perspective and Clinical Outcome.
It is a retrospective study to determine the incidence and outcome of C4d-negative ABMR. Level of evidence III. Introduction:
There are many immunological and non-immunological causes of graft dysfunction, one of the most important factor is rejection either TCMR or ABMR (the last one has a worse prognosis).
When antibodies directed against performed DSA or de novo DSA(HLA or non -HLA antigens) lead to activation of complement cascade and inflammatory cell infiltrates in PTC or glomeruli, and so on C4d stained and become a good marker of complement fixing circulating antibody to endothelium and ABMR. Materials and Methods:
Retrospective study of indicated diagnostic RABs performed from January 2013 to December 2015.
Following serum .creatinine and kidney histological categories were classified as per Revised Banff’13 diagnostic categories for RA biopsies.
C4d- negative ABMR and evaluate their outcome in terms of graft function and survival. Patients of this group were compared to those having C4d-positive ABMR or TCR. Study design:
According to biopsy result patients classified in to three groups
Group 1 :revealed ABMR.
Group 2 combined ACR +ABMR.
Group 3 with ACR.
And Group 1 and Group 2 were further subdivided on the basis of presence (C4d positive) and absence (C4d negative) of C4d deposits on PTC membranes.
Anti-rejection regimen:
Patients who developed cellular rejection were treated with intra- venous methyl prednisolone 500 mg/day for 3–5 days; rabbit anti-thymoglobulin (r-ATG) 1.5 mg/kg BW was added for resistant cases.
C4d-positive ABMR was treated with three sessions of plasmapheresis (with 40 mL/kg BW/session) followed by IVIg 5 g on alternate days and subsequent bortezomib, 1.3 mg/m2 on days 1, 4, 7. and 11 or rituximab, 50–300 mg single dose.
For combined TCR + ABMR, methylprednisolone and rATG were administered followed by plasmapheresis.
C4d-negative rejection were offered monotherapy with either bortezomib or rituximab without plasma exchange. Results & Discussion:
-C4d-positive ABMR (Group 1) presented earlier than other groups and a higher S. creatinine level than any other subgroup.
-Follow up of creatinine reveled that C4d-negative ABMR injury does affect the graft function if not treated aggressively and causes graft attrition in the long-term.
-Graft survival: graft loss appears to be higher in the C4d-positive group as compared to the C4d-negative group.
Recent BANFF classification illustrated two ABMR phenotypes
The first one is mainly due to pre-sensitized recipient which is mainly C4d + and second one who developed de novo DSA which is likely is c4d negative ABMR which has effect on graft survival also.
Some studies have concluded that C4d- negative ABMR tends to occur slightly later post-transplant and has a subclinical presentation with better graft outcome than C4d- positive ABMR.
Graft survival of patients with C4d-negative ABMR was 97.5% as against C4d-positive ABMR which was 95.5% but also has poor graft function. Conclusion:
C4d-positive and C4d-negative ABMR both can occur early or late post-kidney transplant.
Graft outcomes were better in patients who developed C4d-negative ABMR as compared to those who have C4d-positive ABMR.
Molecular studies would be helpful to deter- mine antibodies responsible for C4d-negative ABMR , trying to find targeted therapy to prevent the morbidity associated with this type of injury and improving graft survival .
Banff 13 update consider C4d AMR as a separate intity for allograft dysfunction without a thorough elaboration on clinical and prognostic aspect of the condition.
This is retrospective study conducted in India between 2013 and 2016,
Aim : To determine the incidence and outcome of C4d negative AMR.
Study design:
987 patients presented with renal allograft dysfunction, had allograft biopsy .
categorized into 3 groups :
Group 1 AMR
Group 2 AMR and CMR
Group 3 CMR.
IHC for C4d was performedfor all patients with AMR and mixed AMR and CMR.
Follow up of 3 years post biopsy and treatment.
Results :
Revealed 36% with AMR pure and combined of which C4d negative was 18%.
C4d negative AMR Group showed higher serum creatinine at the end of 3 years of follow up.
C4d negative AMR is less common than C4d positive AMR. And it’s associated with better survival in comparison to C4d positive AMR.
Both can occur in combination with CMR.And both can happened early or late
C4d negative AMR is associated with DSAs against Class II HLA antigens ,they are mostly de novo anti HLA antibodies.
C4d negative AMR is associated with subclinical course and tend to occur slightly later and associated with better prognosis than C4d positive AMR.
the study suggested that a non complement fixing DSAs with antibody dependent cell=mediated cytotoxicity as the main mechanism of AMR.Similarly Autoan5ibodies to the angiotensin II type I receptor have also been associated with graft lossand fibrinoid necrosis resembling AMR but with no PTC C4d .
The study was retrospective study.
Level of evidence is 3
This is retrospective study with level of evidence 3, performed between January 2013 to December 2015 in India. The histological diagnosis was independently reported by a panel of five pathologists and consensus generated was finally reported.
• Kidney biopsy specimens were processed for LM and C4d IHC. Patients whose biopsies revealed immunological rejection were included in the study.
• Patients who underwent ABO incompatible transplants were excluded from the study.
• Histological readings were classified as per Revised Banff’13 diagnostic categories.
• All the biopsies were compared for mean posttransplant and mean follow-up time period in months.
• Patient and graft loss data along with SCr levels were used to determine patient and graft survival.
The biopsies were categorized into three main groups.
• Group 1 pure ABMR
• Group 2 combined ACR +ABMR
• Group 3 pure ACR.
Alloantibodies attack peritubular and glomerular capillaries while T- cells infiltrate tubules and arterial endothelium.
Antibody mediated rejection in group 1 and 2 were further subdivided on the basis of positivity of C4d staining.
Results
404 out of 934 biopsies revealed immunological injury on histology.
• 27.7% biopsies revealed pure ABMR (Group 1)
• 60.6% mixed ABMR + TCR (Group 2)
• 11.3% pure TCR (Group 3).
The detection of C4d is not always indicative of antibody response and C4d negative does not exclude antibody mediated rejection .The incidence of C4d-negative ABMR was less than that of C4d-positive ABMR.
SCr level in C4d-negative ABMR (pure) was higher as compared to the control and other groups.
The overall graft survival appears to be better in the C4d-negative group as compared to the C4d-positive ABMR.
Graft loss appears to be higher in the C4d-positive group as compared to the C4d-negative group.
ABMR has a worse prognosis than T-cell-mediated rejection
The presence of peritubular capillary (PTC) C4d deposition in biopsies is associated with poorer prognosis.
This article focus on C4d negative antibodies mediated rejection and it’s role in outcome of graft survival.
Kidney transplant remain the gold standard therapy in patients with chronic kidney disease.
Kidney allograft rejection is a common problem may lead to graft loss if not treated properly.
There’s Immunological and non immunological factors contribute to graft loss.
So several clinical presentations of allograft rejection hyperacute / accelerated acute / acute and active chronic.
there’s two different mechanisms of allograft rejection are cellular (T cell mediated rejection); Humoral ( B call mediated rejection).
B cell mediated rejection carry worse prognosis than T cell.
Alloantibodies are responsible for acute and chronic kidney transplant rejection.
The pathogenesis of allograft rejection is circulating DSA & de novo DSA which developed from exposure to previous antigen.
de novo DSA directed against MHC class I related chain A antigen and MHC class I related chain B antigen.
Another de novo DSA antibody are angiotensin type 1 receptor antigen and platelets specific antigen. renal biopsy remain the best diagnostic procedure in allograft rejection whereas the presence of C4d along peri tubular capillary and in endothelial cell of glomerulus carry poor prognosis for patients with allograft rejection.
C4d is fragment of C4b developed from activation of classic complement.
C4d is detected easily by immunohistochemistry and indicates for presence of DSA class I and class II antigens binding in endothelial tissue of graft leading to graft injury.
C4b depositions is specific markers and associated with ABMR.
C4d deposits in endothelial tissue for long time even after disappear of Ig and C1 ( it’s footprints along peritubular capillary).
Material and Method:
A retrospective cohort study done in Saudi from January 2013 to December 2015 based on diagnosis of renal allograft rejection on biopsy
Demographic data regarding age gender and indication of renal biopsy and graft function estimated by creatinine level.
renal biopsy done by using variety of staining. Formalin’s fixed biopsy were processed for light microscopy and C4d immunohistochemistry staining by use paraffin.
C4d staining negative biopsy restaining to avoid analytical errors.
Criteria including in this study are patients with immunological rejection and ABO incompatible.
Missing fallow up are excluded.
Reporting of renal allograft biopsy:
Adequate biopsy:
10 nonsclerotic glomeruli and 2 arteries.
Marginal biopsy:
7-9 glomeruli and one artery
Minimal acceptable biopsy
7 glomeruli and one artery
Inadequate biopsy
less than 7 glomeruli and no artery.
Histological categories are classified according to banff 13.
definition of C4d positive ABMR:
1. ABMR defined by presence of circulating DSA along with glomerulitis and peritubular capillarities on histology with C4d deposition across PTC membrane by immunohistochemistry and immunofluorescence.
2. Acute thrombotic microangiopathy whatever cause.
3. Glomerulitis with score g150%
– [ ] 4. Peritubular capillarities with neutrophils and mononuclear cell infiltration
ptc1 10%
Mixed ABMR + TCR with C4d negative / and with positive C4d.
estimation of DSA for screening of anti HLA specific IgG Ab by single antigen bead and Luminex platform and mean fluorescence intensity.
Study design:
All biopsy were compared for mean post transplant and mean fallow up time period in months.
biopsy are divided into three different groups.
gp 1: positive ABMR / gp2: ACR + ABMR / gp 3 ACR
gp 1&2 subdivided into negative C4d and positive C4d PTC membrane.
Anti rejection regime:
All patients with rejection treated by Anti rejection regime.
patients with cellular rejection treated with Iv methyl prednisone
r-ATG add to resistant cases.
C4d positive treat by plasma exchange plus Ig plus bortezomib or rituximab
patients with mixed ABMR & TCR treated by methyl prednisone plus plasma exchange plus r-ATG
patients with C4d negative treated by rituximab or bortezomib.
Results:
Total patients in this study are 987 all biopsied only 5.3% excluded and 43.3% shows immunological injury
27% pure ABMR
60% mixed ABMR with TCR
11.3% pure TCR
C4d negative ABMR was less than c4d positive ABMR patients.
Graft loss:
Mean post transplant period time of allograft biopsy from 5 days to 8 years post transplant .
when biopsy were grouped into those present within one year and those present after one year incidence of c4d positive rejection more than negative c4d
patients with c4d positive have higher serum creatinine levels than those negative c4d.
patients with c4d positive treated aggressive with plasma exchange.
Graft survival: it’s better patients with C4d negative
Graft loss appear higher in patients with C4d positive in comparison with C4d negative
Discussion:
The most cause of ABMR in early post transplant is due to C4d positive.
in late post transplant is due to de novo DSA
C4d negative usually appear after 12 months post transplant but may appear acutely in high sensitised patients.
Autoantibodies like Angitensin type 1&2 receptor are associated with graft loss and fibrinoid necrosis
Incidence of ABMR are high in this study may related to multiple transplant or pre sensitised patients and multiple pregnancies and low hygienic conditions.
TCR usually present early than c4d positive ABMR
finally patients with c4d positive ABMR irrespective with presence or absence of TCR should be treated aggressively by plasma exchange plus r-ATG and Ig.
This article cohort retrospective study
level 3
mai shawky
3 years ago
Q 1.Summary:
· Acute rejection (AR) represents immune mediated graft damage which is associated with worse graft outcome.
· AR may be either :
o AMR ; with histopathological damage mainly in peritubular and glomerular capillaries.
o TCMR: damage mainly by lymphocyte infiltration in tubules, interstitium and arteriolar intima.
· AMR has 2 types;
o Type 1= early post transplant= preformed DSA = mainly c4d+ve.
o Type 2= late post transplant= denovo DSA= mainly c4d -ve.
· No sufficient evidence about relation to c4d positivity to graft outcome, with conflicting results.
· Many studies showed c4d -ve AMR may have better prognosis than c4d +ve. However, C4d-ve AMR still carries a risk of poor long-term graft outcome compared to those without rejection.
· Banff 2013 included c4d -ve AMR.
· C4d meaning:
o It represents activation of complement cascade.
o It remains bound to endothelium at site of injury by covalent bonds, so remain detectable for longer period even after disappearance of antibodies.
o So it represents foot print of AMR.
o It is associated with presence of circulating DSA (detected by Luminex SAB).
· Methods of c4d detection:
o IF (more sensitive), but mostly requires fresh tissue in frozen section 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 However, also IF can be done nowadays on paraffin section by special technique from available blocks.
· General histopathological definitions for biopsy adequacy:
o Adequate = 10 non sclerotic glomeruli+ 2 arteries.
o Marginal = 9 glomeruli+ one artery.
o Minimally accepted= 7 glomeruli+ one artery.
o Unaccepted= 7 glomeruli+ no artery.
· Definitions of positivity of c4d in biopsy:
o Circulating DSA+ intensity ≥ 2 by IF or >0 by IHC.
o C4d +ve may be scored to (1=< 10% of ptc, 2 if between 10-50% and 3 if > 50 % of Ptc involved).
· C4d negative AMR may be due to :
1. Technical errors
2. Different method of c4d detection (IF vs IHC).
3. Very low amount of c4d (difficult detection).
4. Poor complement fixation by DSA
5. Complement independent pathway AMR or antibody dependent cell mediated cytotoxicity.
6. Antibodies against Angiotensin II type 1 receptors
7. fc receptors of Nk may have a role c4d negative AMR.
8. Increased expression of endothelial transcripts causing endothelium injury.
9. Sure pure TCMR will have -ve c4d.however, mixed rejection may be either c4d +ve or -ve.
· C4d +ve staining without AMR:
o Not every c4d +ve means AMR: (sometimes accommodation to antibodies occurs so no immunologically mediated damage occur).
o ABO incompatible transplantation will have +ve c4d.
o Immune mediated kidney disease as lupus nephritis or anti phospholipid syndrome (either recurrent in the graft or infrequently present in donor and not detected). However, mostly here the c4d deposits will be mesangial or glomerular (not in peritubular capillaries).
· C4d -ve AMR in the current study were treated by either rituximab or bortezomib ( no PEX).
· Further longitudinal studies are needed to characterize c4d -ve AMR in relation to prognosis and ideal treatment.
Q2. kind of this study: Retrospective cohort
Q3. level of evidence? Level III
Innocent lule segamwenge
3 years ago
C4d-Negative Antibody-Mediated Rejection: A Pathologist’s Perspective and Clinical Outcome
Please summarise this article
Renal allograft dysfunction (RAD) can be caused by a variety of things. Rejection being one of the common causes. It can be T cell (TCR) or antibody mediated rejection (ABMR) and biopsy is the gold standard for diagnosis. ABMR has a worse prognosis and requires different treatment to TCR. Alloantibodies cause damage to glomerular and peritubular capillaries vs TCR which T cells infiltrate tubules and arterial endothelium. Antibodies can be pre-formed or de novo. Directed against HLA- antigens but can also be directed against non-HLA molecules like MHC Class I related chain A antigens (MICA-A), MHC Class I-related chain B antigens (MICA-B), platelet-specific antigens, renin–angiotensin pathway molecules. C4d deposition in peritubular capillaries is associated with presence of DSA and caries a poor prognosis. C4d is a fragment of complement C4b a product of the classic complement pathway. C4d remains bound to tissues for several days. It is a footprint of antibody response. Antibodies cause allograft injury through complement activation. May also upregulate complement regulatory protection and cytoprotective proteins leading to accommodation. In this study renal allograft biopsies were reviewed to determine the incidence of ABMR and determine outcomes in terms of graft function and survival. A retrospective study of renal allograft biopsies between m January 2013 to December 2015 Biopsies consistent with immunological rejection were included in the study ABO incompatible transplants and patients lost for follow- up were excluded from the study C4d positive ABMR was defined by presence of glomerulitis and peritubular capillaritis and C4d + staining in peritubular capillaries. C4d negative ABMR was defined as above except for C4d <2 on immunofluorescence. TCR- defined as tubulitis t>1,along with vasculitis (v>0) and/or interstitial inflammation (i>2). DSA was determined by single antigen bead testing. 987 biopsies analysed. 5.3% excluded. Remained 934 biopsies. 404/934 (43.3%) had evidence of immunological injury. 27.7% biopsies revealed pure ABMR (group1) 60.6% mixed ABMR + TCR (group 2) 11.3% pure TCR ( group 3) C4d-negative rejection was 18.48% in Group 1 and 18.7% in Group 2 Most of the rejections occurred in males 80.4%. Times of biopsy ranged from 5 day to 8 years post-transplantation. C4d+ ABMR presented earlier 1.32 ± 0.28 months Followed by mixed ABMR+TCR There were C4d+ than C4d – in the study. C4d+ ABMR were more likely to have high serum creatinine than C4d -. The mean HLA match was better in C4d+ patients (2.2) compared to C4d- (1.5) C4d negative injury if untreated causes graft loss in the long run. C4d negative had better graft survival compared to C4d+ ABMR o graft loss reported in the C4d-negative ABMR Conclusion C4d negative causes graft injury if left untreated, however presence of C4d positive injury leads to more severe graft injury and loss of graft function.
What is the kind of this study?
A descriptive retrospective study
What is the level of evidence?
Level 3 evidence
Amit Sharma
3 years ago
I. C4d-Negative Antibody-Mediated Rejection: A Pathologist’s Perspective and Clinical Outcome
Please summarise this article
Graft dysfunction is a common scenario post- kidney transplant. Kidney rejection may be either hyperacute, accelerated acute, acute or chronic active rejection.
Rejection, which can be classified as either cellular or antibody mediated rejection (AMR), has major impact on graft prognosis. Acute AMR is seen in 5-7% of transplant recipients and makes up for 20-48% of all acute rejections. Antibodies in AMR act on peritubular and glomerular capillaries, while the T cells act on tubules and arterial endothelium in T cell mediated rejection.
C4d, formed by the classical complement pathway, gets attached to endothelial cell surface by sulfhydryl group. Although C4d detection is an indirect sign of antibody mediated injury, there is a group of AMR patients who are C4d negative.
This was a retrospective study (from January 2013 to December 2015) analysing the C4d negative AMR and comparing it with C4d positive AMR. The study included all graft biopsies done for indication showing rejection except those with ABO incompatible transplants and those who did not have adequate follow-up. Formalin-fixed kidney biopsies were assessed for light microscopic changes and C4d immunohistochemistry (IHC).
Standard definitions for adequacy of biopsy, parameters for glomerulitis, peritubular capillaritis, C4d scoring, C4d positive AMR, C4d negative AMR, T cell mediated rejection (TCR), and combined AMR with T cell mediated rejection were used.
Donor specific antibodies (DSA) were estimated using single antigen bead testing and normalized MFI >1000 was taken as positive.
C4d positive AMR: Circulating DSA with biopsy showing glomerulitis, peritubular capillaritis and C4d>0 on IHC.
C4d negative AMR: Circulating DSA with biopsy showing glomerulitis, peritubular capillaritis and C4d0 on IHC.
TCR: Biopsy with tubulitis (t>1), vasculitis (v>0) and interstitial inflammation (i≥2).
AMR with TCR: Combination of TCR and g≥1.
All the cases were divided into 3 groups. Group 1 included AMR, further divided into1a (C4d positive) and 1b (C4d negative).Group 2 included combined MAR and TCR, further divided into 2a (C4d positive) and 2b (C4d negative).Group 3 included TCR.
Patients with TCR were treated with methylprednisolone, and ATG if resistant.Patients with C4d positive AMR were treated with Plasmapheresis, IVIG, bortezomib or rituximab. For C4d negative patients, patients were given either rituximab or bortezomib without plasmapheresis. Combined AMR and TCR was treated with methylprednisolone and ATG.
Demographic profile, serum creatinine, HLA matching and graft survival was analysed.
Out of 987 biopsies, 404 (43.3%) had rejection, majority of whom were males. 27.7% of them had pure AMR, 60.6% had combined MAR and TCR while 11.3% had TCR alone. C4d negative AMR was less common with approximately 18% in both group 1 and group 2. Mean serum creatinine at time of biopsy was higher in C4d positive AMR. DSA was present in all patients with AMR. Mean HLA match in C4d negative group was lower than in the C4d positive group. Serum creatinine levels on follow-up were highest in C4d negative AMR group, probably due to the type of treatment given in this group.
Graft survival was better in C4d negative group with higher graft loss in C4d positive group.
AMR has 2 major phenotypes: Phenotype 1 is seen early, in presensitized patients and C4d positive while phenotype 2 is seen later, is due to de novo DSA and likely C4d negative, due to class II HLA antibodies, causing late graft loss.
C4d negative AMR is usually seen more than 1-year post-transplant, but can be seen early in highly sensitized patients, has subclinical presentation with better prognosis than C4d positive AMR. C4d negative AMR, in association with TCR, can present earlier than C4d positive AMR.
The limitation of the study included lack of re-biopsy in C4d negative AMR to look for any change in C4d stain status, as a different group had shown that C4d negative AMR can become C4d positive.
The study concluded that C4d negative AMR incidence is low, with better graft survival but poorer graft function as compared to C4d positive AMR.
Dear All
Review the level of evidence. Follow this diagram
Please summarise this article
Kidney transplantation is the treatment of choice for ESRD patients. However, the allograft might be endangered by rejection episodes, of which ABMR is the worst. ABMR accounts for 20-48% of all rejection episodes.
According to Banff classification, ABMR could be diagnosed by evidence of tissue injury (microvascular inflammation), evidence of antibody reaction (c4d staining) and serological tests detecting DSA. However, ABMR may be diagnosed in absence of C4d staining and called c4d negative ABMR.
–This is a retrospective study that included 987 transplant recipient graft biopsies between 2013 and 2015. The specimens were stained for H&E, and c4d immunohistochemistry.
The histopathological findings were defined as
1- C4d positive ABMR: glomerulitis, DSA and c4d staining score more than 0 by IHC, and more than 2 by IF
2- C4d negative ABMR: C4d <2 on IF, acute TMA in the absence of any definitive cause.
they concluded:
– low incident value for c4d negative rejection.
-No specific risk factors could be detected as causes for c4d negative ABMR.
-Timing for c4d negative was comparable to that for C4d positive rejection, both early and late.C4d negative ABMR had a better outcome than positive.
What is the kind of this study?
Retrospective cohort study
What is the level of evidence?
III
This is a retrospective study from January 2013 to December 2015 in India to illustrate the incidence and outcome of C4d-negative ABMR with level 3 evidence. Allograft proven immunological rejection were included. Recipients who underwent ABO incompatible transplants and defaulted follow- up were excluded. Allograft biopsies with immunological injuries classified as Group 1: pure Antibody Mediated Rejection ( ABMR ), Group 2: combined ABMR with concurrent T-cell-mediated rejection (TCR), and Group 3: pure TCR. Groups 1 and 2 again subdivided into subgrouped as C4d positive (Group 1a and 2a) or C4d negative (Group 1b and 2b). Graft function was measured by serum creatinine. Out Of the 987 allograft biopsies, 43.3% (404) of biopsies revealed immunological injury. Of these, 27.7% of the biopsies revealed pure ABMR (Group 1), 60.6% revealed combined ABMR with TCR (Group 2), and 11.3% revealed pure TCR (Group 3). The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%. The C4d-negative rejections were 18.48% and 18.7% respectively in Group 1 and Group 2. The mean serum creatine were higher in c4d negative rejection at the end of three years follow-up. In conclusion, C4d-negative ABMR has a low incidence, presents early after transplantation but carries better outcome than C4d-positive ABMR. However, further trial with longer duration is needed.
Summary
C4d detection can be called a footprint of antibody response. Although this product does not have any functional role, it serves as evidence of humoral rejection of graft for transplant teams.
C4d forms an important part of diagnostic measures for rejection, with treatment protocols including a combination of plasmapheresis, IVIG, immunoadsorption, Tacrolimus/mycophenolate mofetil, and Rituximab.
Banff criteria for ABMR and its types are the focus of this paper.
C4d negative ABMR has better chances of graft survival but poor graft function reflected by higher serum creatinine level.
In conclusion, this paper relies on data post transplant to show that C4d negative ABMR is to be taken as seriously as C4d positive ABMR, and treated aggressively irrespective of absence of TCR.
Type of study : Retrospective study
Level of evidence : 3
Level of evidence 3
Biopsy still remains the gold standard and provides valuable insights intothepathogenesis ofearly and late allograft injury
Alloantibodies preferentially attack the peritubular and glomerular capillaries in contrast to Tcells which characteristically infiltrate tubules and arterial endothelium
C4b/C4d remains bound in the tissuefor several daysafter Ig and C1 have been released.
Covalent binding renders C4d, a stable moleculeto be easily detected by immunohistochemistry
Detection of C4d is regarded as “footprint” of an antibody response and is currently the best single marker of complementfixing circulating antibodies to the endothelium .
is currently the best single marker of complement fixing circulating antibodies to the endothelium
The presence of DSAs within the graft does not necessarily mean activation of thecomplement, pathological changes, and graftdysfunction, since accommodation may occur
twomajorphenotypes of ABMR. The first type appears early in the posttransplant period in apresensitizedpatient and is more likely to be C4dpositive.
The second type develops late posttransplant and is due to de novo DSA development and islikely to be C4dnegative.
C4d Negative subset of patients also needs tobe treated aggressively like those with C4d-positive ABMR irrespective of concurrent or absence of TCR.
Level of evidence III
Retrospective cohort study.
-Renal transplantation (RT) is a well-established therapy for patients with end-stage renal disease. Renal allograft dysfunction (RAD) is a common and complex problem of RT. Appropriate management is critical for longterm graft function and survival.
– ABMR generally has a worse prog- nosis and requires a different form of therapy
than T-cell-mediated rejection (TCR).
-ABMR is caused by antibodies directed against donor-specific human leukocyte antigen (HLA) molecules or other cell antigens. The most common
mechanism underlying ABMR is an amnestic robust antibody response that originates from previous antigenic exposure or de novo development of DSA.
-C4d is a fragment of complement C4b, an activation product of the classic complement
pathway. C4b (and C4d) contains an occult sulfhydryl group that forms a covalent, thio- ester bond with nearby proteins on activation by antibody and C1. C4b/C4d remains bound in the tissue for several days after Ig and C1 have been released.
-Detection of C4d is regarded as an indirect sign, a “footprint” of an antibody response.
-Definitions Adequacy – Optimal biopsy was defined as aspecimen with at least 10 nonsclerotic glomeruli and two arteries, a marginal biopsy having seven to nine glomeruli and one artery and a minimally acceptable biopsy having seven
glomeruli and one artery. Specimens with <7 glomeruli or no arteries or with only medulla were considered as inadequate for interpretation . Histological categories were classified as per Revised Banff’13 diagnostic categories for RA biopsies.
-The incidence of C4d-negative ABMR is strikingly low in our study. The demographic
profile or clinical symptoms are not helpful in determining the risk factors for the
development of C4d-negative ABMR. In our study, C4d-positive and C4d-negative ABMR
shows similar frequencies of concurrent cell- mediated rejection and both can occur early or late posttransplant. Graft outcomes were com- paratively better in the cohort of patients who developed C4d-negative ABMR as compared to those who have C4d-positive ABMR. However, longitudinal prospective studies are required to know the clinical significance and long-term outcome of C4d-negative ABMR. Molecular studies would be helpful to deter- mine antibodies responsible for C4d-negative ABMR to design targeted therapy approach to this group so as to prevent the morbidity associated with this type of injury and impro- ving graft survival in this cohort of patients.
• Renal allograft biopsy is the gold standard for diagnosis allograft dysfunction. Acute rejection (AR)has several types: hyper acute, accelerate acute, acute and chronic active rejection with cellular and /or cellular and humoral immunological pathways. AR determines allograft survival and its long-term outcome. ABMR is responsible for 20-48% of ARs and has worse prognosis and requires specific treatment. Donor specific antibodies (DSAs)can be performed before TX, or be de novo which are produced after TX and discovered 1960s and 1970s respectively. DSA can be anti-HLA class I and II Ags or against non-HLA Ags such as M1CA, M1CB. C4d has a covalent binding to endothelial cell surfaces and remain near the sites of C4 activation B and easily detected by IHC foot print of complement fixing Ab response and endothelial injury. High-titer DSAS cause ABMR and low titer can develop accommodation.
• This retrospective study reviewed allograft biopsies to find incidence of C4d-negative ABMR and compare their outcome with C4d positive ones. Renal biopsy was reported by five pathologists and a final report was generated. Variables such as age, gender, indication of biopsy and serum creatinine were collected. Biopsy specimens were assessed by light microscopy and C4d IHC. Luminex was performed and MFI>1000 was considered positive. Three group of biopsy were identified: 1-ABMR 2- ABMR+ ACR 3- ACR (a)C4d+ and (b)C4d- were subgroup for group 1 & 2. Patients were treated according their biopsy results.
TCR received methylprednisolone 3-5 days and then ATG if resistant.
ABMR(C4d+): plasmapheresis (40 mg/kg /session) and 5 gr IVIG (alternate days) then bortezomib (1.3 mg/m2) on days 1,4,7,11 or rituximab.
Combined TCR+ABMR: methylprednisolone and ATG then plasmapheresis.
Outcome was determined by graft loss.
• Results: of 934 allograft biopsies, 404 cases showed injury. ABMR was shown in 27.7% of biopsies (group D). Mixed ABMR +TCR (group 2) in 60.6% and pure TCR in 11.3% of cases. C4d negative rejection was shown in 18.48% and 18.7% in group 1& 2.
• The incidence of C4d negative (group1b) was 4.7% in males and 0-4.9 % in females and for group2b was 1.98% in females and 9.4% in males. There was more C4d +rejection comparing to C4d – ABMR in this study. DSA was detected in all patients with ABMR and none of TCR cases. Better HLA matching was associated with better graft survival. Graft survival was better in C4d –group comparing C4d+ group, although mean Scr was higher in this group. There was no graft loss in pure C4d- ABMR group. They concluded that C4d- ABMR has low incidence, early presentation and better outcome than C4d –positive AMBR. Pre sensitized patients were more likely C4d+ where as those who developed donor DSA were more likely C4d-.
• This is a retrospective study with level of Evidence of 3.
Introduction
ABM rejections occur in 5-7 % of renal transplants and 20 % -48 % of acute rejections
usually may be due to Pre formed DSA, or Denovo DSA against Class I, II HLA Antigen
or antibodies against the non-HLA antigen ( MICA, MICB antigens, platelet-specific antigens ACE pathway and chemokines and its receptors )
C4d positive biopsies associated with poor prognosis and is considered an excellent marker for the presence of antibodies directed against allograft kidneys.
C4d positive ABM rejection is defined as :
presence of :
– DSA
-Glomerulitis and peritubular capillaritis
– C4d positive ≥ 2 in IF staining or >0 with IHC staining
C4d negative ABM rejection defined as :
presence of :
-DSA
–Glomerulitis peritubular capillaritis (g >0 and PTC >0 or g +PTC ≥ 2 or g>0 or PTC>0 )in addition to TMA with absence of etiology)
C4d positive <2 in IF staining or =0 with IHC staining
Study Design :
All biopsies were compared with follow-up of creatinine in patients
and divided into 3 groups :
GroupI :ABM rejection
Group II : ACR + ABM rejections
Group III: ACR
with groups, I and II subdivided into subgroups C4d positive or C4d negative
Rejections treatment:
ACR : methyl prednisolone 500 mg/day for 3–5 days + ATG for resistant cases with dose 1.5 mg/Kg
C4d positive ABMR: Plasma exchange 3sessions + IVIG 5 gm on alternate days + either
bortezomib with dose of 1.3 mg/meter square for 1,4, 7 , 11 days
or Rituximab 500 mg single dose
Combined ACR + ABM : methyl prednisolone + ATG + plasma exchange
Outcome :
Biopsies of pure ABMR: 27.7 %
Biopsies of Mixed rejections : 60.6 %
Biopsies of Pure ACR: 11.3 %
C4d negative 18.4 in group I ,18.7 % in group II
with graft survival better in the C4d negative group compared to the C4d positive group
Limitations :
No rebiopsy was done during the follow up with the possibility of conversion of C4d negative to positive
No quantitative DSA done
Discussion:
ABMR of two phenotypes :
Phenotype I early post-transplant in pre sensitized patient
Phenotype II late post-transplant : due to De novo DSA
C4d negative ABMR tends to be late posttransplant with better graft survival than C4d positive
Endothelial injury expression is usually higher in ABM rejections
C4d negative less frequently than c4d positive
Conclusion :
C4d positive and C4d negative have the same frequencies with cell-mediated rejections with poorer graft survival and can occur early or late.
Graft survival is better in C4d negative ABMR than in C4d positive ABMR
What is the kind of this study?
Retrospective Study.
What is the level of evidence?
Level III
Acute rejection (AR) episodes are a major determinant of renal allograft survival and long term outcome of renal transplant (RT). Acute antibody–mediated rejection (ABMR) occurs in 5%–7% of all RTs and is responsible for 20%–48% of AR episodes. The prevalence of chronic ABMR
varies from 5% at one year to 20% at five years. ABMR generally has a worse prognosis and requires a different form of therapy than T-cell-mediated rejection (TCR).
Antibodies directed against donor-specific human leukocyte antigen (HLA) molecules or other cell antigens. The most common mechanism underlying ABMR.
peritubular capillary (PTC) C4d deposition in RT biopsies is strongly associated with a poor prognosis and raises the possibility that antibodies are responsible for graft loss.
C4d is a fragment of complement C4b, an activation product of the classic complement pathway.
Detection of C4d is regarded as an indirect sign, a “footprint” of an antibody response. ABMR presents most of the time as severe allograft dysfunction and its prompt diagnosis and optimal
treatment are essential.
Materials and Methods
an Institutional Review Board approved retrospective study of indicated diagnostic RABs performed from January 2013 to December 2015.
A panel of five pathologists independently reported.
The demographic data regarding patient age, gender, indication for RA biopsy, and serum
creatinine were collected. Graft function was measured by SCr levels (mg/dL).
Formalin-fixed biopsy specimens were processed for light microscopy and C4d immunohistochemistry as per manufacturer’s protocol.
For light microscopy, 3 μm thick sections were stained with hematoxylin and Eosin, Gomori’s
trichrome, periodic acid–Schiff, and Jones silver methenamine stains.
Inclusion and exclusion criteria
Patients whose biopsies revealed immunological rejection were included in the study.
Patients who underwent ABO incompatible transplants and those who were lost for follow up were excluded from the study.
Reporting of renal allograft biopsy
Definitions:
Adequacy – Optimal biopsy was defined as a specimen with at least 10 non-sclerotic glomeruli and two arteries, a marginal biopsy having seven to nine glomeruli and one artery and a
minimally acceptable biopsy having seven glomeruli and one artery.
Histological categories were classified as per Revised Banff’13 diagnostic categories for RA biopsies.
Definition of C4d-positive ABMR – ABMR was defined if there was the presence of
circulating DSA along with glomerulitis and peritubular capillaritis on histopathology with
the presence of C4d deposition across the PTC membranes with an intensity of ≥2+ on
immunofluorescence and C4d >0 by immunohistochemistry on paraffin-embedded sections.
Acute thrombotic microangiopathy (TMA) in the absence of any other cause was also reported
as ABMR
Glomerulitis (g>0) – Glomerulitis was defined as the percentage of glomeruli that revealed infiltration by leukocytes in the capillaries.
Score g1, g2, and g3 were offered if <25%, 26%–50%, and >50% of glomeruli showed leukocytic infiltration, respectively.
Peritubular capillaritis – The percentage of PTCs in the renal cortex that contained neutrophils or mononuclear cells was defined as peritubular capillaritis. A score of ptc1 was offered if <10% PTCs revealed cells, ptc2 when 10% of PTCs were infiltrated by <5 cells, and ptc3 if more than 10% of PTCs revealed >10 cells/PTC
C4d scoring – Demonstration of PTC C4d deposition by immunohistochemistry was considered positive if grade was >C4d0. Score of C4d1, C4d2, and C4d3 was given if 1–9%, 10%–50%, and >50% PTCs were involved, respectively.
C4dnegative ABMR was defined as biopsies with C4d <2 on immunofluorescence or C4d >0 on
IHC, ptc> 0 and g >0, g+ptc ≥2, or g>0 or ptc >0 and acute TMA in the absence of any other cause of TMA.
Definition of TCR – TCR was defined as biopsies with tubulitis (t>1) along with vasculitis (v>0) and/or interstitial inflammation (i>2).
Study design
All the biopsies were compared for mean post-transplant and mean follow-up time period in months.
SCr levels (mg/dL) at the time of biopsy and at last follow-up were compared.
Patient and graft loss data along with SCr levels were used to determine patient and graft survival.
The biopsies were categorized into three main groups.
Group 1 comprised of biopsies that revealed ABMR,
Group 2 combined ACR +ABMR,
and Group 3 with ACR.
Group 1 and Group 2 were further subdivided on the basis of presence
(C4d positive) and absence (C4d negative) of C4d deposits on PTC membranes.
Anti-rejection regimen
All the patients who developed rejection were subjected to antirejection regimen which was decided according to the type of rejection reported on biopsies.
Patients who developed cellular rejection were treated with intravenous methyl prednisolone 500 mg/day for 3–5 days; rabbit anti-thymoglobulin (r-ATG) 1.5 mg/kg BW was added for resistant cases.
C4d-positive ABMR was treated with three sessions of plasmapheresis (with 40 mL/kg BW/session) followed by i.v. Ig 5 g on alternate days and subsequent bortezomib, 1.3 mg/m2 on days 1, 4, 7. and 11 or rituximab, 50–300 mg single dose.
For combined TCR + ABMR, methylprednisolone and rATG (in doses mentioned above) were administered followed by plasmapheresis.
Patients with C4d-negative rejection were offered monotherapy with either bortezomib or rituximab.
None of these patients underwent therapeutic plasma exchange.
Outcome
The death-censored graft loss was determined by the time between the date of transplantation and either date of graft failure indicated by need for dialysis, date of death, or last date of
follow-up with a functioning graft.
Patient death or dialysis dependency was the proposed end points of this study. We compared the death censored graft loss against each subgroup.
Conclusion
The incidence of C4d-negative ABMR is strikingly low in this study.
What is the kind of this study?
· Cohort retrospective study
· What is the level of evidence?
· Level 3.
1- Summarize tis article
Alloantibodies are now considered important cause of acute and chronic rejection, differing in pathogenesis from TCR.
Alloantibodies preferentially attack a different “location,” namely the peritubular and glomerular capillaries in contrast to T cells which characteristically infiltrate tubules and arterial endothelium.
ABMR is caused by antibodies directed against (HLA) molecules or other cell antigens.
Antibodies can be directed against other donor-specific antigens such as
· MHC Class Irelated chain A antigens,
· MHC Class I-related chain B antigens,
· platelet-specific antigens,
· molecules of the renin–angiotensin pathway,and
· polymorphisms involving chemokines and their receptors.
C4d is fragment of C4 complement .It is produced by activation of classical complement pathway. C4d is a footprint of antibody response .
C4d deposition is strongly associated with circulating antibody to donor HLA Class I/II antigens and is cur rently the best single marker of complement fixing circulating antibodies to the endothe lium.
staining of allograft biopsies for the fragment C4d is a specific and reliable method for identifying lesions due to allo-antibodies against HLA Class I and/or Class II antigens.
Nearly 20%–30% of all Rejection episodes have a humoral component.
C4d staining of graft biopsies has emerged as an important tool for the diagnosis
of antibody-dependent allograft injury independent from its occurrence early or late after transplantation.
Antibodies can mediate endothelial injury through complement-dependent and independent mechanisms.
High-titer DSAs may cause ABMR, while recipients with low-titer DSAs can develop accommodation.
The presence of DSAs within the graft does not necessarily mean activation of the complement, pathological changes, and graft dysfunction, since accommodation may occur
In this study the incidence of C4d-negative ABMR was less than that of C4d-positive ABMR.
The most recent Banff meeting update highlights two major phenotypes of ABMR.
The first type appears early in the posttransplant period in a presensitized patient and is more likely to be C4d positive.
The second type develops late posttransplant and is due to de novo DSA development and is likely to be C4d negative. The second phenotype is an important factor in late graft loss.
patients.
Class II HLA molecules may be responsible and that much of the endothelial damage is mediated by NK cells and, monocytes and neutrophils; antibody-dependent cell-mediated cytooxicity.
C4d-negative ABMR usually occurs >12 months after transplantation but can occur acutely in highly sensitized patients with persistent DSAs .
Causes of negative C4d negative .
1- Technical issues related to type of fixative used,
2- different methods of C4d detection,
3- poor complement fixation by some DSAs,
4- Comple ment- independent pathway of ABMR
Studies have concluded that C4d negative ABMR tends to occur slightly later posttransplant and has a subclinical presen tation with better graft outcome than C4d positive ABMR.
Graft outcomes were comparatively better in patients whith C4d-negative ABMR compared to C4d-positive ABMR
2-What is the kind of this study?
This is retrospective study
3-What is the level of evidence?
Level 3
Banff’13 update included C4d-antibody-mediated rejection (ABMR) as a separate entity responsible for graft dysfunction with limited clinical/prognostic implications.
This was a retrospective study which reviewed Renal allograft biopsies done between jan2013 till jan2016. A panel of five pathologists independently reported and consensus diag- nosis generated was finally reported.
Patients whose biopsies revealed immuno- logical rejection were included in the study. Patients who underwent ABO incompatible transplants and those who were lost for follow- up were excluded from the study.
Study design
All the biopsies were compared for mean posttransplant and mean follow-up time period in months. SCr levels (mg/dL) at the time of biopsy and at last follow-up were compared. Patient and graft loss data along with SCr levels were used to determine patient and graft survival. After analyzing the light microscopic features, the biopsies were categorized into three main groups. Group 1 comprised of biopsies that revealed ABMR, Group 2 combined ACR +ABMR, and Group 3 with ACR. Group 1 and Group 2 were further subdivided on the basis of presence (C4d positive) and absence (C4d negative) of C4d deposits on PTC membranes. Group 1a and Group 2a included biopsies with PTCs that demonstrated C4d deposition (C4d posi- tive) and Group 1b and Group 2b included biopsies that revealed the absence of C4d (C4d negative) on PTC membranes.
Anti-rejection regimen
All the patients who developed rejection
were subjected to antirejection regimen which
was decided according to the type of rejection
reported on biopsies. Patients who developed
cellular rejection were treated with intra-
venous methyl prednisolone 500 mg/day for
3–5 days; rabbit anti-thymoglobulin (r-ATG)
1.5 mg/kg BW was added for resistant cases.
C4d-positive ABMR was treated with three
sessions of plasmapheresis (with 40 mL/kg
BW/session) followed by i.v. Ig 5 g on alter-
nate days and subsequent bortezomib, 1.3
2
mg/m on days 1, 4, 7. and 11 or rituximab,
50–300 mg single dose. For combined TCR + ABMR, methylprednisolone and rATG (in doses mentioned above) were administered followed by plasmapheresis. Patients with C4d-negative rejection were offered mono- therapy with either bortezomib or rituximab. None of these patients underwent therapeutic plasma exchange.
Outcome
The death-censored graft loss was determined by the time between the date of transplantation and either date of graft failure indicated by need for dialysis, date of death, or last date of follow-up with a functioning graft. Patient death or dialysis dependency was the proposed end points of this study. We compared the death- censored graft loss against each subgroup.
A total of 987 RA biopsies from 987 patients
43
were analyzed; 5.3% biopsies were excluded from the study. Of the remaining 934 allograft biopsies, 43.3% (404) biopsies revealed immunological injury on histology. Out of these, 27.7% biopsies revealed pure ABMR (Group 1), 60.6% mixed ABMR + TCR (Group 2), and 11.3% pure TCR (Group 3). The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%. The incidence of C4d-negative rejections was 18.48% and 18.7% in Group 1 and Group 2, respectively. The incidence of C4d-negative ABMR was less than that of C4d-positive ABMR.
The incidence of C4d-negative ABMR is strikingly low in this study. The demographic profile or clinical symptoms are not helpful in determining the risk factors for the development of C4d-negative ABMR. In this study, C4d-positive and C4d-negative ABMR shows similar frequencies of concurrent cell- mediated rejection and both can occur early or late posttransplant. Graft outcomes were comparatively better in the cohort of patients who developed C4d-negative ABMR as compared to those who have C4d-positive ABMR.
I. C4d-Negative Antibody-Mediated Rejection: A Pathologist’s Perspective and Clinical Outcome
# Please summarise this article
# Introduction
* Renal transplantation (RT) is best solution for patients with end stage renal disease.
* Renal allograft rejection is major problem of RT.
*Types of rejection that can occur including hyperacute, accelerated acute, acute, and chronic active rejections.
*The immunologic pathways involved in rejection divided into cellular (T cell mediated) and humoral (antibody/B cell mediated) pathways.
* There are a lot of diagnostic methodologies for diagnosis of rejection but biopsy still remains the gold standard method for detection early and late allograft injury.
*ABMR generally has a worse prognosis and requires a different form of therapy than (TCR).
*Alloantibodies are important mediators of acute and chronic rejection it attack the peritubular and glomerular capillaries, while Tcells infiltrate tubules and arterial endothelium.
*ABMR is caused by antibodies directed against donor-specific human leukocyte antigen (HLA) molecules or other cell antigens that originates from previous antigenic exposure or de novo development of DSA.
* Study showed that peritubular capillary (PTC) C4d deposition in RT biopsies is strongly associated with a poor prognosis and raises the possibility that antibodies are responsible for graft loss.
* Feucht et al suggested that capillary C4d deposition was an evidence for humoral alloreactivity against the graft and strongly associated with circulating antibody to donor HLA Class I/II antigens and best single marker of complement fixing circulating antibodies to the endothelium.
# Materials and Methods
This was retrospective study performed from January 2013 to December 2015.
*Data (patient age, gender, indication for RA biopsy, and serum creatinine were collected)
*Formalin-fixed biopsy specimens were processed for light microscopy and C4d immunohistochemistry
*Inclusion and exclusion criteria Patients with biopsies revealed immunological rejection were included in the study.
Patients with ABO incompatible or were lost for follow up excluded from the study.
* C4d positive ABMR was defined if there was the presence of circulating DSA along with glomerulitis and peritubular capillaritis on histopathology with the presence of C4d deposition across the PTC membranes with an intensity of ≥2+ on immunofluorescence and C4d >0 by immunohistochemistry on paraffin-embedded sections.
* C4dnegative ABMR was defined as biopsies with C4d 0 on IHC, ptc> 0 and g >0, g+ptc ≥2, or g>0 or ptc >0
* TCR was defined as biopsies with tubulitis (t>1) along with vasculitis (v>0) and/or interstitial inflammation (i>2).
*Definition of ABMR with TCR biopsies
with C4d-negative ABMR which also had
histological evidence of TCR also had g ≥1
*Detection of the DSA by si ngle antigen bead for HLA Class I and II
any normalized MFI value over 1000 was considered positive.
# Study design
*After analyzing the light microscopic features, the biopsies were categorized into three main groups. Group 1 revealed ABMR,
Group 2 combined ACR +ABMR and Group 3 with ACR.
* Group 1 and Group 2 were subdivided to Group 1a and 2a included biopsies with PTCs that demonstrated C4d deposition (C4d positive) and Group 1b and 2b included biopsies that revealed the absence of C4d (C4d negative) on PTC membranes.
# Anti-rejection regimen
Patients who developed cellular rejection were treated with IV methyl prednisolone 500 mg/day for (3–5 days) and r-ATG 1.5 mg/kg BW was added for resistant cases.
C4d-positive ABMR was treated with 3 sessions of plasmapheresis followed by i.v. Ig 5 g on alternate days and subsequent bortezomib, 1.3 mg/m2 on days 1, 4, 7. and 11 or rituximab 50–300 mg single dose.
For combined TCR + ABMR, methylprednisolone and rATG followed by plasmapheresis.
Patients with C4d-negative rejection with bortezomib or rituximab.
# Results
*Out of 934 allograft biopsies, 43.3% of biopsies revealed immunological injury on histology.
*27.7% revealed pure ABMR (Group 1).
*60.6% mixed ABMR + TCR (Group 2).
*11.3% pure TCR (Group 3).
* The overall incidence of ABMR (pure ABMR+ ABMR with TCR) was 36.27%.
* The incidence of C4d-negative rejections was 18.48% and 18.7% in Group 1 and Group 2, respectively.
*The incidence of C4d-negative ABMR was less than that of C4d-positive ABMR.
# Discussion
*The most recent Banff update highlights two phenotypes of ABMR. The first type appears early in the posttransplant in a presensitized patient and it is C4d positive the second type develops late posttransplant and is due to de novo DSA development and it is C4d negative.
*It appears that Class II HLA molecules may be responsible of the endothelial damage is mediated by NK cells, monocytes and neutrophils; antibody-dependent cell-mediated cytooxicity.
* C4d-negative ABMR usually occurs >12 months after transplantation but can occur acutely in highly sensitized patients with persistent DSAs
*Technical issues related to some causes of C4d negativity. *Autoantibodies to the angiotensin II type 1 receptor also
associated with graft loss and fibrinoid
necrosis similar to ABMR except for the absence of
PTC C4d.
* Fc receptors on NK cells (FcRIIA) also play a role in AR.
*There is significant proportion in ABMR transit between negative C4d staining and positive C4d deposition in the peritubular capillaries.
*C4d negative ABMR occur later posttransplant and has a subclinical presentation with better graft outcome than C4dpositive ABMR.
*Sis et al reported that expression of endothelial cellassociated
transcripts (ENDATs) is present in all types of rejection but higher in ABMR.
*When C4d negative ABMR occurs alone it presents later than C4d-positive ABMR but if concurrent TCR is also seen, it presents earlier than C4d-positive ABMR.
*Study concluded that C4d-positive and C4d-negative ABMR show similar frequencies of concurrent cell mediated rejection and both can occur early or late posttransplant.
*The mean SCr levels at the time of presentation was low in the C4d-negative ABMR, but the mean SCr over the mean follow-up period appears to be higher inC4d-negative ABMR compared to other groups these may be attributed to not subjecting these patients to aggressive management.
*Although the C4d negative ABMR associated with better graft survival,
it is poorer graft function reflected by the higher serum creatinine indicating the immune injury so this patients needs to be treated aggressively like those with C4d-positive ABMR irrespective of concurrent or absence of TCR.
# What is the kind of this study?
Retrospective cohort study
#What is the level of evidence?
Level of evidence is level 3
This study is retrospective observational study with level 3 of evidence.
This study evaluates incidence and outcomes of C4d negative AMR in 987 kidney transplant recipients.
Renal allograft dysfunction can be result from ABMR and/or TCMR. Based on this article, several types of rejection including hyperacute, accelerated acute, acute, and chronic active rejection have been described. Biopsy is gold standard for evaluation of kidney dysfunction and allograft rejection in kidney transplant recipients.
Acute rejection has a deleterious effect on graft survival and ABMR is responsible for 20-48% of AR. Incidence chronic ABMR is reported between 5 -20%. ABMR has worse prognosis compared to TCMR. Formation of de novo DSA has detrimental effect on graft survival.
Alloantibodies preferentially attack peritubular and glomerular capillaries, but T cells typically infiltrate tubules and arterial endothelium.
The most common mechanisms for ABMR is amnestic robust response to previously exposed antigens and de novo DSA formation. Antibodies direct against HLA class I and class II, MICA, MICB, PLT-specific antigens, molecules of renin-angiotensin pathway, and chemokines and its receptors.
C4d is a fragment of C4b and is marker of activation of classical pathway and it remains on the endothelial cell surfaces, vascular basement membrane, and in intracytoplasmic vacuoles of endothelial cells. C4d positive staining is footprint of antibody response. C4d staining is the best marker of complement fixing antibodies to the endothelium and is an important tool for the diagnosis antibody mediated graft injury.
By upregulation of complement regulatory and inhibitory pathways, under certain conditions, antibodies can lead to graft accommodation. Low titer of DSAs can induce accommodation. Thus, presence of DSAs do not always mean activation of complement and graft injury.
Early diagnosis of ABMR is crucial because it needs heavy treatment with IVIG, plasmapheresis, immunoadsorption, and Rituximab. Two different phenotypes of ABMR were classified in Banff 2013. The first type occurs early post-transplant and in pre-sensitized patients, and more likely to be C4d positive. The second type is due to de novo DSA and present late post-transplant and more likely to be C4d negative. It is important in late graft loss, HLA class II molecules are more probably responsible, and it is more plausibly due to NK cell mediated endothelial injury. C4d negative ABMR usually occurs after 1 year post transplantation but can present in highly sensitized recipients.
Different fixation techniques, different methods of DSA detection, DSA with poor complement fixation or complement independent pathways such as anti-angiotensin II type I antibodies are causes of C4 negative staining. The FcRIIA on NK cells mediated graft injury can be another cause of C4d negativity.
Transition between C4d positive staining and C4d negative staining in ABMR is reported. C4d negative ABMR occur later post-transplant with subclinical presentation and better graft outcomes compared to C4 positive ABMR. Although ENDATs is present in all types of rejection, it presents higher in ABMR especially in C4d negative ABMR.
One study reported C4d positive and C4d negative ABMR had similar frequency of concurrent TCMR.
In this study, the overall incidence of C4d negative ABMR was 18.2 %, significantly less than C4d positive ABMR. It was correlated with later presentation but in concurrence with TCMR occurred earlier.
In C4d negative group, although serum Cr level was lower in the beginning of study, it was higher at the end of mean follow up period. The overall graft survival was 97.5% and 95.5% in C4d negative and C4d positive, respectively.
C4d negative ABMR should be treated similar to C4d positive ABMR with no hesitation.
Introduction
Types of Renal allograft rejection: Hyperacute, acute, and chronic by two immunological pathways humoral and cellular.
ABM rejections occur in 5-7 % of renal transplants and 20 % -48 % of acute rejections
usually, alloantibodies directed against peritubular and glomerular capillaries
etiology of ABM rejections: Presenistised DSA, or Denovo DSA against Class I, II HLA Antigen
or antibodies against the non-HLA antigen ( MICA, MICB antigens, platelet-specific antigens ACE pathway and chemokines and its receptors )
The poor prognosis of ABM rejections especially if associated with C4d positive biopsies
C4d has no functional role but it is considered to be an excellent marker for the presence of antibodies directed against allograft kidneys.
with an endothelial injury can be mediated either complement-dependent or not.
higher DSA leads to ABM and lower DSA leads to accommodation.
C4d positive ABM rejection is defined as :
presence of circulating DSA + glomerulitis and peritubular capillaritis + C4d positive ≥ 2 in IF staining or >0 with IHC staining
C4d negative ABM rejection defined as :
presence of circulating DSA + (g >0 and PTC >0 or g +PTC ≥ 2 or g>0 or PTC>0 in addition to TMA with absence of etiology) + C4d positive <2 in IF staining or =0 with IHC staining
Study Design :
All biopsies were compared with follow-up of creatinine in patients
and divided into 3 groups :
GroupI :ABM rejection
Group II : ACR + ABM rejections
Group III: ACR
with groups, I and II subdivided into subgroups C4d positive or C4d negative
Rejections treatment:
ACR : methyl prednisolone 500 mg/day for 3–5 days + ATG for resistant cases with dose 1.5 mg/Kg
C4d positive ABMR: Plasma exchange 3sessions + IVIG 5 gm on alternate days + either
bortezomib with dose of 1.3 mg/meter square for 1,4, 7 , 11 days
or Rituximab 500 mg single dose
Combined ACR + ABM : methyl prednisolone + ATG + plasma exchange
Outcome :
Biopsies of pure ABMR: 27.7 %
Biopsies of Mixed rejections : 60.6 %
Biopsies of Pure ACR: 11.3 %
C4d negative 18.4 in group I ,18.7 % in group II
with graft survival better in the C4d negative group compared to the C4d positive group
Limitations :
No rebiopsy was done during the follow up with the possibility of conversion of C4d negative to positive
No quantitative DSA done
Discussion:
ABMR of two phenotypes :
Phenotype I early post-transplant in pre sensitized patient
Phenotype II late post-transplant : due to De novo DSA
C4d negative ABMR tends to be late posttransplant with better graft survival than C4d positive
Endothelial injury expression is usually higher in ABM rejections
C4d negative less frequently than c4d positive
Conclusion :
C4d positive and C4d negative have the same frequencies with cell-mediated rejections with poorer graft survival and can occur early or late.
Graft survival is better in C4d negative ABMR than in C4d positive ABMR
What is the kind of this study?
Retrospective Study.
What is the level of evidence?
Level III
Please summarise this article:
Introduction
Acute antibody–mediated rejection (ABMR) occurs in 5%–7% of all RTs and is responsible for 20%–48% of AR episodes. ABMR generally has a worse prognosis and requires a different form of therapy than T-cell-mediated rejection (TCR ),C4d is a fragment of complement C4b, an activation product of the classic complement pathway.
Detection of C4d is regarded as an indirect sign, a “footprint” of an antibody response.
C4d deposition is strongly associated with circulating antibody to donor HLA Class I/II antigens and is currently the best single marker of complement fixing circulating antibody.
Materials and Methods:
This was an Institutional Review Board approved retrospective study of indicated
diagnostic performed from January 2013 to December 2015,( ABO incompatible transplants and those who were lost for follow- up were excluded from the study),
C4d- negative ABMR was defined as biopsies with C4d <2 on immunofluorescence or C4d >0 on IHC, ptc> 0 and g >0, g+ptc ≥2, or g>0 or ptc >0 and acute TMA in the absence of any other cause of TMA.
Aim of study: a retrospective study to determine the incidence and outcome of C4d-negative ABMR.
Results and discussion:
A total of 987 RA biopsies from 987 patients were analyzed,the overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%. The incidence of C4d-negative rejections was 18.48% and 18.7% in Group 1 and Group 2, respectively. The incidence of C4d-negative ABMR was less than that of C4d-positive ABMR.
All the patients who revealed immunological injury were subjected to qualitative DSA estimation and we found a 100% positivity in patients having ABMR. DSA was absent in all the cases with TCR,C4d-negative injury does affect the graft function if not treated aggressively and causes graft attrition in the long-term.
The overall graft survival appears to be better in the C4d-negative group as compared to the C4d-positive ABMR group in spite of the higher mean SCr levels in this group.
The overall incidence of ABMR was higher in our study. This could be attributed to various causes such as transplantation across HLA barriers, multiple transfusions, presensitized patients, and multiple pregnancies in women apart from low hygienic conditions prevailing in this country.
The overall incidence of C4d-negative ABMR in our study was 18.2%, which is much less than C4d-positive ABMR (72.3%). C4d–negative ABMR when occurs alone, usually presents later than C4d-positive ABMR (1.51 vs. 1.32 months).
Limitations :
study cohort with C4d-negative ABMR was not re-biopsied during follow-up. Hence, the possibility of C4d negative converting to C4d positive could not be determined. we did not perform quantitative DSA estimation due to financial constraints and compare with biopsy results.
CONCLUSION:
C4d-positive and C4d-negative ABMR shows similar frequencies of concurrent cell- mediated rejection and both can occur early or late posttransplant. Graft outcomes were comparatively better in tpatients who developed C4d-negative ABMR as compared to those who have C4d-positive ABMR.
What is the kind of this study?
This is retrospective study
What is the level of evidence?
Level 3
This is a retrospective study (level III)
A total of 987 renal allografts were investigated. c4d positive AMBR was considered in case of 2 or more intensity for IF and >0 for IHC in addition to the classical definition of the existence of DSA and glomerulitis and capillaritis. presence of acute TMA was considered as AMBR. histological categories were defined according to revise BANFF13. biopsies were divided in 3 groups (group one that revealed acute AMBR, group to ACR and ABMR, group 3 ACR. group 1 and 2 were subdivided into either c4d positive or negative.
27% revealed pure ABMR (group1)
60.6% had mixed ABMR and TCR (group 2)
11 % had pure TCR (group 3)
the overall incidence of AMR either pure or cıomined was 36.27 %
the incidence of C4d-negative was 18.48% and 18.7 in group1 and 2 respectively. this is less than c4d positive ABMR
a limitation of this study is suspicious incidence of c4d because c4d negatives were not rebiopsied (the probability of being positive later is not excluded). DSA levels were quantified.
retrospective study to determine the incidence and outcome of C4d-negative ABMR.
Sample size 987 renal allograft (RA) biopsies obtained from 987 RA recipients were studied from January 2013 to January 2016.
All samples were subjected to light microscopy using standard stains and C4d immunohistochemistry on paraffin sections and reported according to modified Banff’s criteria.
Adequate biopsies with immunological injuries were categorized as
· Group 1: pure ABMR
· Group 2: combined ABMR with concurrent T-cell-mediated rejection (TCR)
· Group 3: pure TCR.
· Groups 1 and 2 were further subgrouped as C4d positive (Group 1a and 2a) or C4d negative (Group 1b and 2b).
43.3% (404) biopsies revealed immunological injury. Of these,
· 27.7% of the biopsies revealed pure ABMR (Group 1),
· 60.6% revealed combined ABMR with TCR (Group 2)
· 11.3% revealed pure TCR (Group 3).
· The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%, of which C4d-negative rejections were 18.48% and 18.7% in Group 1 and Group 2
The mean SCr at the end of three years follow-up in patients with C4d-negative rejections was comparatively higher. C4d-negative ABMR, recently included in Banff’13, has a low incidence, usually presents early after transplantation but carries better outcome than C4d-positive ABMR. However, further long term studies are still required for knowing the clinical course over years.
methodology
excellent study design in which diagnostic biopsies performed from January 2013 to December 2015.
A panel of five pathologists independently reported and consensus diagnosis generated was finally reported to decrease the human error in the results.
The demographic data regarding patient age, gender, indication for RA biopsy, and serum creatinine were collected. Graft function was measured by SCr levels (mg/dL).
Formalin-fixed biopsy specimens were processed for light microscopy and C4d immuno-histochemistry as per manufacturer’s protocol. For light microscopy, 3 μm thick sections were stained with hematoxylin and Eosin, Gomori’s trichrome, periodic acid–Schiff, and Jones silver methenamine stains. The C4d staining was performed on 3 μm thick paraffin sections. Biopsies negative for C4d were re-stained to rule out analytical errors associated with staining procedure. Membranous/lupus nephropathy slides were used as positive controls. Endothelial cells lining the medium caliber blood vessels were taken as internal control.
Inclusion and exclusion criteria
· Patients whose biopsies revealed immunological rejection were included in the study.
· Patients who underwent ABO incompatible transplants and those who were lost for follow- up were excluded from the study.
Reporting of renal allograft biopsy Definitions Adequacy
· Optimal biopsy was defined as a specimen with at least 10 non sclerotic glomeruli and two arteries
· a marginal biopsy having seven to nine glomeruli and one artery
· a minimally acceptable biopsy having seven glomeruli and one artery.
Specimens with <7 glomeruli or no arteries or with only medulla were considered as inadequate for interpretation.
Definition of C4d-negative ABMR – C4d- negative ABMR was defined as biopsies with C4d <2 on immunofluorescence or C4d >0 on IHC, ptc> 0 and g >0, g+ptc ≥2, or g>0 or ptc >0 and acute TMA in the absence of any other cause of TMA.
Definition of TCR – TCR was defined as biopsies with tubulitis (t>1) along with vasculitis (v>0) and/or interstitial inflammation (i>2).
Definition of ABMR with TCR – Biopsies with C4d-negative ABMR which also had histological evidence of TCR also had g ≥1 so as to qualify the criteria of ABMR.
Quantitative estimation of DSA by single antigen (SA) testing – HLA Class I and Class II antibody specificity screening was performed with Single Antigen Beads on Luminex platform following the manufacturer’s protocol. Trimmed mean fluorescence intensity (MFI) values were obtaine the flow analyzer and normalized using the formula: [(Sample #N beads – Sample negative control (NC) beads) – (NC serum #N beads – NC serum NC beads)]. Any normalized MFI value over 1000 was considered positive.
Limitations
· cohort with C4d-negative ABMR was not re-biopsied during follow-up. Hence, the possibility of C4d negative converting to C4d positive could not be determined.
· We did not perform quantitative DSA estimation due to financial constraints and compare with biopsy results.
· the mean SCr over the mean follow-up period appears to be higher in Group 1b (C4d-negative ABMR) when compared to other groups Treatment regimens were different in both groups (c4d +ve and c4d-ve)>> c4d +ve received more aggressive treatment.
Conclusion
· The incidence of C4d-negative ABMR is low in study.
· The demographic profile or clinical symptoms are not helpful in determining the risk factors for the development of C4d-negative ABMR
· C4d-positive and C4d-negative ABMR shows similar frequencies of concurrent cell- mediated rejection and both can occur early or late posttransplant.
· Graft outcomes were comparatively better in the cohort of patients who developed C4d-negative ABMR as compared to those who have C4d-positive ABMR. However, longitudinal prospective studies are required to know the clinical significance and long-term outcome of C4d-negative ABMR.
· Molecular studies would be helpful to determine antibodies responsible for C4d-negative ABMR to design targeted therapy approach to this group so as to prevent the morbidity associated with this type of injury and improving graft survival in this cohort of patients.
type of study retrospective observetional study
level of evidence 3
Please summarise this article
What is the kind of this study?
What is the level of evidence?
This is a retrospective study; class 3 evidence.
Summary:
Renal transplantation is the modality of choice for patients with ESKD. Different immunological pathways can cause rejection as cellular or humoral. Graft biopsy is considered as the gold standard method for rejection diagnosis.
Acute ABMR may occur in 5%-7% of all recipients.
The study included 934 graft biopsies. All recipients with evidence of immunological rejection were included in the study. Recipients with ABO incompatible transplantation & those who lost follow-up were excluded.
Adequate-optimal biopsy :at least 10 glomeruli & 2 arteries.
Marginal biopsy :at least 7-9 glomeruli + 1 artery.
Minimal accepted :biopsy at least 7 glomeruli + 1 artery.
C4d positive ABMR means the presence of DSA with glomerulitis and peritubular capilliritis in addition to positive staining of C4d across PTC membrane.
C4d negative ABMR means presence of DSA with glomerulitis and peritubular capilliritis with C4d staining <2 by IF.
TCR means the presence of tubulitis, vascular inflammation with or without interstitial inflammation.
All patients are treated for rejection according to the result of biopsy with pulse steroid, ATG for the TCR or PTx and IVIg followed by bortizumib & rituximab for the ABMR .
Outcome:
-End point of study was patient death or dialysis dependency.
C4d negative ABMR may be due to:
-Technical errors
-poor complement fixation by DSA
-complement independent pathway ABMR
-antibodies against ACEII type 1 receptors
-NK Fc receptors may have a role in acute rejection with C4d negative staining.
It was conclude that the ABMR with C4d negative is less frequent and associated with better prognosis than ABMR C4d positive but still carries a poor graft survival.
Retrospective study
Level of evidence 3
C4d- negative ABMR:
• is less common than C4d- positive
• occurs more late (usually after one year)
• less severe than C4d- positive
Retrospective study
Level 3 evidence
Conducted from to 2013 to 2016
Study 987 renal allograft biopsies to determine the incidence and outcome of C4d negative ABMR
They divided the patients to 3 groups
1-pure ABMR
2- combined ABMR and TCR
3-pure TCR
Then divided group 1&2 to sub group C4d positive and C4d negative (by IP to paraffin sections) they excluded those undergo ABO incompatible Transplant (5.3%)
So the result
43% had immunological reaction,27% of them revealed pure ABMR ,60% mixed rejection and 11% pure TCR
So the incidence of C4d negative ABMR was low and the graft survival was better in these group
Introduction ;
Material and Methods ;
Reporting of renal allograft biopsy ;
Definitions
Anti-rejection therapy ;
2.C4d-positive ABMR ;
3.ABMR + TCMR ;
4.C4d negative ABMR ;
Outcome ;
Statistical analysis
Results ;
Graft survival ;
Discussion ;
Limitations ;
Conclusions ;
Retrospective study
Level 3
Thank You
C4d-Negative Antibody-Mediated Rejection: A Pathologist’s Perspective and Clinical Outcome
☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆☆
▪︎This was an Institutional Review Board approved retrospective study of indicated diagnostic Renal Allograft Biopsies (RABs) performed from January 2013 to December 2015.
☆Introduction:
________________
▪︎ Biopsy is the gold standard for diagnosis of renal allograft dysfunction (RAD)
▪︎Alloantibodies are important mediators of acute and chronic rejection, differing in pathogenesis or “nature,” from T.cell mediated rejection (TCR).
▪︎ Alloantibodies preferentially attack a different “location,” namely the peritubular
and glomerular capillaries in contrast to Tcells which characteristically infiltrate tubules and arterial endothelium.
▪︎ABMR is caused by antibodies directed against donor-specific HLA molecules.
▪︎AR associated with the development of de novo anti-HLA DSA in recipient’s serum is a defined clinicopathologic entity carrying a poor prognosis and
▪︎Complement-neutrophil PW is the major mechanism of antibody injury.” And neutrophils in peritubular and glomerular capillaries are strongly associated with circulating anti-donor HLA antibodies.
▪︎Peritubular capillary (PTC) C4d deposition in RT biopsies is strongly associated with a poor prognosis and raises the possibility that antibodies are responsible for graft loss.
▪︎ C4d is an activation product of the C4. C4b (and C4d) contains an occult sulfhydryl group that forms a covalent, thioester bond with nearby proteins on activation by antibody and C1. C4b/C4d remains bound in the tissue for several days after Ig and C1 have been released.
▪︎Following activation C4d bind to endothelial cell surfaces and extracellular matrix components of vascular basement membranes near the sites of C4 activation.
▪︎C4d is also found in intracytoplasmic vacuoles of endothelial cells. Covalent binding renders IH.
▪︎Detection of C4d is regarded as an indirect sign, a “footprint” of an antibody response.
▪︎ No functional role of C4d per se has been reported.
▪︎C4d deposition is strongly associated with circulating antibody to donor HLA Class I/II antigens and is currently the best single marker of complement fixing circulating antibodies to the endothelium.
▪︎Capillary C4d deposition was an evidence for humoral alloreactivity against the graft.
▪︎Staining of allograft biopsies for the fragment C4d is a specific and reliable method for identifying lesions due to allo-antibodies against HLA Class I and/or Class II antigens.
▪︎ C4d staining of RA biopsies is an important tool for the diagnosis of antibody-dependent allograft injury independent from its occurrence early or late
after transplantation.
▪︎Antibodies can mediate endothelial injury through complement-dependent and independent mechanisms.
▪︎ Under certain conditions, antibodies can lead to graft accommodation (in low titre DSA)
☆Materials and Methods:
_________________________
▪︎ This is a retrospective study which determine the incidence and outcome of C4d-negative ABMR.
▪︎ It studied 987 renal allograft (RA) biopsies obtained from Jan 2013 to Jan 2016 and subjected to light microscopy using standard stains (IHC)on paraffin sections and reported according to modified Banff’s criteria.
▪︎Adequate biopsies with immunological injuries were categorized as:
Group 1: pure ABMR,
Group 2: combined ABMR with concurrent
T-cell-mediated rejection (TCR)
Group 3: pure TCR.
▪︎Groups 1 and 2 were further subgrouped as C4d positive (Group 1a and 2a) or C4d
negative (Group 1b and 2b).
▪︎ Graft function was measured by serum creatinine (SCr) level (mg/dL).
▪︎ 43.3% of biopsies revealed immunological injury. Of these, 27.7% of the biopsies revealed pure ABMR (Group 1), 60.6% revealed combined ABMR with
TCR (Group 2), and 11.3% revealed pure TCR (Group 3).
▪︎The overall incidence of ABMR (pureABMR + ABMR with TCR) was 36.27%, ofwhich C4d-negative rejections were 18.48% and 18.7% in Group 1 and Group 2, respectively.
▪︎The mean SCr at the end of three years follow-up in patients with C4d-negative rejections was comparatively higher.
☆Discussion:
______________
▪︎C4d staining is an important part of the diagnostic workup in suspected cases of ABMR.
▪︎ The most recent Banff meeting update highlights two major phenotypes of ABMR.
1) Appears early in the posttransplant period in a presensitized patient and is more likely to be C4d positive.
2) Develops late posttransplant and is due to de novo DSA development and is likely to be C4d negative (Class II HLA molecules may be responsible).
▪︎C4d-negative ABMR usually occurs >12 months after transplantation but can occur
acutely in highly sensitized patients with persistent DSAs (even after desensitization).
▪︎ Causes of C4d negativity:
1) Technical issues.
2) Different methods of C4d detection
3) Poor complement fixation by some DSAs
4) Complement-independent PW of ABMR
5) Autoantibodies to the angiotensin II type 1 receptor
6) AR due to Fc receptors on NK cells (FcRIIA)
▪︎C4dnegative ABMR tends to occur slightly later posttransplant and has a subclinical presentation with better graft outcome than C4d positive ABMR.
▪︎ C4d-negative ABMR is much less than C4d-positive ABMR (72.3%).
▪︎ C4d–negative ABMR when occurs alone, usually presents later than C4d-positive ABMR; however, if concurrent TCR is also seen then it usually presents earlier than
C4d-positive ABMR.
▪︎ The overall graft survival is better in C4d-negative ABMR group than the cohort of
C4d-positive ABMR
▪︎C4d-negative ABMR does have a poorer graft function as reflected by the higher
serum creatinine levels.
▪︎There is a subset of patients also needs to be treated aggressively like those with C4d-positive ABMR irrespective of concurrent or absence of TCR.
▪︎ There is a possibility of C4d negative converting to C4d positive
☆Conclusion:
_______________
▪︎The demographic profile or clinical symptoms are not helpful in determining the risk factors for the development of C4d-negative ABMR.
▪︎C4d-positive and C4d-negative ABMR shows similar frequencies of concurrent cell mediated rejection and both can occur early or late posttransplant.
▪︎ Graft outcomes were comparatively better in C4d-negative ABMR as compared
to C4d-positive ABMR
▪︎ Longitudinal prospective studies are required to know the clinical significance and long-term outcome of C4d-negative ABMR.
▪︎Molecular studies would be helpful to determine antibodies responsible for C4d-negative ABMR.
__________________
☆What is the kind of this study?
Retrospective cohort study
☆What is the level of evidence? Level 3
Thank You
· Please summarise this article A retrospective study ,
· Jan 2013 to Dec 2015 , 987 transplanted patients underwent renal allograft biopsies, under LM and C4dstaining. S.creatinine was done at time of biopsy . Flow up rage from 5 days to 8 years.
· Inclusion Criteria: Evidence of rejection in allograft biopsies.
Exclusion Criteria: ABO-incompatible transplant and patient lost their follow up.
Adequate biopsies with immunological injuries were categorized
C4d positive AMR: Circulating DSA with biopsy showing glomerulitis, peritubular capillaritis and C4d>0 on IHC.
C4d negative AMR: Circulating DSA with biopsy showing glomerulitis, peritubular capillaritis and C4d 0 on IHC.
TCR : Biopsy with tubulitis (t>1), vasculitis (v>0) and interstitial inflammation (i≥2).
AMR with TCR: Combination of TCR and g≥1.
Outcomes: death censored graft loss (primary endpoint), patient death or dialysis dependency (secondary endpoint)
.Results:
· 43% of the biopsies revealed an immunological injury
27% of showed pure ABMR.
60% combined TCMR+ABMR.
11% pure TCMR.
18% of ABMR were C4d negative
Limitations:
1. No biopsy flow up done for C4d negative patients
2. No DSA level flow up done
3.
Conclusion:
The incidence of C4d negative ABMR is low compare to C4d positive ABMR
Graft outcomes were better in C4d negative ABMR patients.
·
· What is the kind of this study?
· Cohort retrospective study
· What is the level of evidence?
· Level 3
Thank You
This is a level III retrospective research to investigate the incidence and outcome of C4d-negative ABMR in renal allograft biopsies from 987 renal transplant patients.
A total of 987 renal allograft biopsies from 987 renal transplant recipients were analyzed.
Using light microscopy and C4d immunohistochemistry on paraffin sections, all renal allograft biopsy samples were investigated and reported using modified Banff’s criteria for renal allograft biopsy samples. The findings of the renal transplant biopsy were separated into groups. Group 1 contains just ABMR, Group 2 has both ABMR and concurrent T-cell-mediated rejection (TCR), and Group 3 contains only TCR. Groups 1 and 2 were further separated into C4d positive (Groups 1a and 2a) and C4d negative (Groups 1b and 2b) individuals (Groups 1b and 2b). The amount of serum creatinine (SCr) (milligrams per deciliter of blood) was used to assess graft function.
Although the demographic profile and clinical symptoms are important, they are not useful in identifying the risk factors for developing C4d-negative ABMR.
Pre-biopsy serum creatinine levels were as follows:
When the serum creatinine levels of each cohort were compared, it was shown that patients who tested positive for pure C4d had a higher SCr level (2.74 0.35) than patients in any other subgroup.
Estimation of donor-specific antibodies in a qualitative manner:
Patients with ABMR test positive 100 per cent of the time. DSA was not present in any of the cases involving TCR.
Match with human leukocyte antigen:
In all groups, the better the HLA match, the greater the transplant survival rate was seen.
The following are the current serum creatinine levels:
The SCr levels in patients with C4d– positive ABMR were lower than those in patients with C4d– negative ABMR because the C4d– positive group received more intensive plasmapheresis treatment than the C4d– negative group. This demonstrates that if C4d-negative damage is not treated quickly, it might have a deleterious impact on graft function and even cause graft attrition over the long term.
Graft survival is a matter of degree.
When comparing the C4d-positive and C4d-negative groups, it appears that graft loss is greater in the C4d-positive group (5.2 per cent vs. 2.5 per cent).
C4d-negative However, in highly sensitized individuals with chronic DSAs, ABMR may manifest themselves within 12 months of transplantation, rather than over time (even after desensitization).
The following are the causes of C4d negativity:
Technical concerns relating to the type of fixative employed led to the employment of several techniques of C4d detection.
Some DSAs have poor complement fixation, and the ABMR route is not dependent on complement fixation.
Autoantibodies to the angiotensin II type 1 receptor have also been linked to graft loss and fibrinoid necrosis, which are similar to alloantibody-mediated rejection except for the lack of PTC C4d, which is frequent in alloantibody-mediated rejection.
Results: The total incidence of ABMR (pure ABMR plus ABMR with TCR) was 36.27 per cent, with C4d-negative rejections accounting for 18.48 per cent and 18.7 per cent in Groups 1 and 2, respectively. Conclusions: Patients with C4d-negative rejections had a significantly higher mean SCr at the end of three years of follow-up than those with C4d-positive rejections. In Banff’13, C4d-negative ABMR was listed for the first time. It has a low incidence and generally manifests very early after transplantation, but it has a better result than C4d-positive ABMR.
level of evidence 3, retrospective cohort study
Thank You
-The recognition of the transplanted organ as foreign is mediated by complex
immunologic pathways, which can be divided into cellular (T-cell mediated) and humoral (antibody/B cell-mediated) pathways.
-biopsy is the gold standard and provides valuable insights into the pathogenesis of early and late allograft injury.
– Acute rejection (AR) episodes are a major determinant of renal allograft (RA )survival and the long-term outcome of renal transplant (RT).
– Acute antibody-mediated rejection (ABMR) occurs in 5%–7% of all RTs and is responsible for 20%–48% of AR episodes.
-The prevalence of chronic ABMR varies from 5% at one year to 20% at five
years.
-ABMR generally has a worse prognosis .
-The most common mechanism underlying ABMR is antibody response that originates from previous antigenic exposure or de novo development of DSA. Other implicated causes could be the presence of antibodies that are directed against HLA/major histocompatibility complex (MHC) class I and II antigens. Antibodies can be directed against other donor-specific antigens such as MHC Class I related chain A antigens, MHC Class I-related chain B antigens, platelet-specific antigens, molecules of the renin–angiotensin pathway, and polymorphisms involving chemokines and their receptors.
-C4d is a fragment of complement C4b, an activation product of the classic complementpathway. Detection of C4d is regarded as an indirect sign, a “footprint” of an antibody response.
-No functional role of C4d has been reported.
-C4d deposition is strongly associated with circulating antibody to donor HLA Class I/II antigens and is currently the best single marker of complement-fixing
circulating antibodies to the endothelium.
-It is a retrospective study performed from January 2013 to December 2015.
-A total of 987 renal allografts (RA) biopsies obtained from 987 RA recipients were studied.
-Graft function was measured by SCr levels (mg/dL).
– Formalin-fixed biopsy specimens were processed for light microscopy and C4d immunohistochemistry as per the manufacturer’s protocol.
-Biopsies negative for C4d were re-stained to rule out analytical errors associated with the staining procedure.
-Optimal biopsy was defined as a specimen with at least 10 nonsclerotic glomeruli and two arteries, a marginal biopsy having seven to nine glomeruli and one artery, and a minimally acceptable biopsy having seven glomeruli and one artery.
-Definition of C4d-positive ABMR – ABMR was defined if there was the presence of circulating DSA along with glomerulitis and peritubular capillaritis on histopathology with the presence of C4d deposition across the PTC membranes with an intensity of ≥2+ on immunofluorescence and C4d >0 by immunohistochemistry on paraffin-embedded sections.
-Definition of C4d-negative ABMR – C4dnegative ABMR was defined as biopsies with C4d <2 on immunofluorescence or C4d >0 on IHC, ptc> 0 and g >0, g+ptc ≥2, or g>0 or PTC >0 and acute TMA in the absence of any other cause of TMA.
-The most recent Banff highlights two major phenotypes of ABMR. The
first type appears early in the posttransplant period in a presensitized patient and is more likely to be C4d positive. The second type develops late posttransplant and is due to de novo DSA development and is likely to be C4d negative. The second phenotype is an important factor in late graft loss.
– C4d-negative ABMR usually occurs >12 months after transplantation but can occur acutely in highly sensitized patients with persistent DSAs (even after desensitization).
-Autoantibodies to the angiotensin II type 1 receptor have also been associated with graft loss and fibrinoid necrosis that resembles alloantibody-mediated
rejection except for the common absence of PTC C4d.
-Several tudies have concluded that C4d-negative ABMR tends to occur slightly later post transplant and has a subclinical presentation with better graft outcome than C4dpositive ABMR.
-The overall incidence of ABMR was higher in this study that could be attributed to various causes such as transplantation across HLA barriers, multiple transfusions, presensitized patients, and multiple pregnancies in women apart from low hygienic conditions.
-The overall incidence of C4d-negative ABMR in this study was 18.2%, which is much less than C4d-positive ABMR (72.3%). C4d–negative ABMR when occurs alone, usually presents later than C4d-positive ABMR (1.51 vs. 1.32 months) .
-The overall graft survival of patients with C4d-negative ABMR was 97.5% as againstC4d-positive ABMR which was 95.5%. Although associated with better graft survival, the C4d-negative ABMR does have a poorer graft function as reflected by the higher serum creatinine levels.
What is the kind of this study?
Retrospective study
What is the level of evidence?
3
Thank You
Summary:
Introduction:
Kidney transplant is the best renal replacement therapy for patients with end stage kidney disease associated with better Quality of life, improved graft survival and overall patient survival. kidney graft dysfunction can be triggered by many immunological and non-immulogical factors ,immunological triggers are common and complex including humoral and cellular immune response which can be lead to different types of allograft rejections ranging from hyper-acute rejection , accelerated rejection , acute and chronic active rejections , graft biopsy still considered as the best method for the diagnosis of the different types allograft dysfunction.
Aim of the study:
·To measure the frequency of c4d negative ABMR and the overall graft function and graft survival as a final outcome.
Method and materials:
·this study reviewing the histopathology of 987 renal allograft biopsies retrospectively including LM and C4D staining by using IHC from paraffin block, Biopsies negative for c4d were restrained to avoid technical error
· study period from Jan, 2013- dec,2016.
They divided the cases in 3 main groups, group one pure ABMR, group 2 mixed rejection (ABMR, ACR), Group 3pure ACR ,Group 2 according to c4d staining they further subdivided in two groups g1a,1b c4d+ve and c4d negative 2a,2b, 5 pathologist independently reviewed the graft biopsies , based on the modified Banff criteria updated in 2013 for the classification of graft injury , membranous and SLE slides used as positive controls , they include all specimens with confirmed allograft injury including acute TMA , 5.3% inadequate biopsies were excluded for interpretations, also they excluded those with ABOI transplantation , and those who lost for follow up.
Antirejection protocols documented according to the type of rejection
Pure ACR, treated with PMP 3-5 Days, ATG in resistant ACR 1.5MG /kg
Pure C4D +VE ABMR (PP, IVIG, plus Bortezomib 1.3mg/kg in 4 doses or single dose rituximab
Mixed rejection (PMP, ATG, PP)
C4D-ve ABMR treated with monotherapy either bortezomib or rituximab, no PP
Results:
43.3% biopsies showed immunological injury, 27.7% pure ABMR
Mixed rejection 60%, 11.3% pure ACR, overall ABMR (PURE, MIXED) was 36.27%
While c4 negative ABMR was lower than C4D+ve ABMR only 18.4and 18.6in group1,2
Majority of patients are young male adults with median age of 33
Mean post – transplant FU period in months was inconstant between the 3 groups with shortest in C4D+ve ABMR group1 , followed by C4D-ve ABMR ,TCR Group2b, and even after one year fu they found that c4d +ve ABMR is predominate type 75.5% compared to 20% of c4d-ve ABMR.Pure C4D positive ABMR more predominate and more sever acute graft dysfunction at time of presentation and up on last FU however C4 -VE ABMR was higher compared to the control group , DSA 100% positive in ABMR
Graft survival outcome:
Graft survival better in C4D-ve despite higher mean SCR level in this group and received less aggressive therapy compared to C4D +ve ABMR
Discussion:
The updated Banff classification highlighted two major phenotypes of ABMR
-phenotype 1 C4D +VE ABMR early post transplantation period in highly sensitized patients
– Phenotype 2 C4D -ve ABMR, late post-transplant, due to denovo anti HLA class11 antibodies and associated with late graft loss but again can occur acutely in highly sensitized patients with persistent DSA despite desensitization, complement independent direct endothelial damage by antibody dependent cell mediated cytotoxicity including NK, neutrophils. IN this cohort group the incidence of ABMR is higher than TCR with C4D -ve ABMR only 18% while C4D +VE predominates of total 72%, this can be explained by higher rate of presensitization with blood transfusion, pregnancies, nonadherence. The prevalence C4D negative ABMR reported similar rate or even higher in some studies as high as 49% (34,33).
Although C4D -ve ABMR shows better graft survival rate in this study but its overall associated with progressive graft loss over time.
Limitations:
Retrospective design
No biopsies for the C4D negative ABMR on FU
Quantitative DSA not available
Type of the study
retrospective cohort study with FU outcome
level of evidence :
2B
sorry i meant 3B
Yes, level 3
Thanks
Aim of the study:
· To measure the frequency of c4d negative ABMR and the overall graft function and survival as an outcome.
Method and materials:
· this study reviewing the histopathology of 987 renal allograft biopsies retrospectively including LM and C4D staining by using IHC from paraffin block, Biopsies negative for c4d were restrained to avoid technical error
· study period from Jan, 2013- dec,2016.
They divided the cases in 3 main groups, group one pure ABMR, group 2 mixed rejection (ABMR, ACR), Group 3pure ACR ,Group 2 according to c4d staining they further subdivided in two groups g1a,1b c4d+ve and c4d negative 2a,2b, 5 pathologist independently reviewed the graft biopsies , based on the modified Banff criteria updated in 2013 for the classification of graft injury , membranous and SLE slides used as positive controls , they include all specimens with confirmed allograft injury including acute TMA , 5.3% inadequate biopsies were excluded for interpretations, also they excluded those with ABOI transplantation , and those who lost for follow up.
Antirejection protocols documented according to the type of rejection
Pure ACR, treated with PMP 3-5 Days, ATG in resistant ACR 1.5MG /kg
Pure C4D +VE ABMR (PP, IVIG, plus Bortezomib 1.3mg/kg in 4 doses or single dose rituximab
Mixed rejection (PMP, ATG, PP)
C4D-ve ABMR treated with monotherapy either bortezomib or rituximab, no PP
Results:
43.3% biopsies showed immunological injury, 27.7% pure ABMR
Mixed rejection 60%, 11.3% pure ACR, overall ABMR (PURE, MIXED) was 36.27%
While c4 negative ABMR was lower than C4D+ve ABMR only 18.4and 18.6in group1,2
Majority of patients are young male adults with median age of 33
Mean post – transplant FU period in months was inconstant between the 3 groups with shortest in C4D+ve ABMR group1 , followed by C4D-ve ABMR ,TCR Group2b, and even after one year fu they found that c4d +ve ABMR is predominate type 75.5% compared to 20% of c4d-ve ABMR
Pure C4D positive ABMR more predominate and more sever acute graft dysfunction at time of presentation and up on last FU however C4 -VE ABMR was higher compared to the control group ,
DSA 100% positive in ABMR
Graft survival outcome:
Graft survival better in C4D-ve despite higher mean SCR level in this group and received less aggressive therapy compared to C4D +ve ABMR
Discussion:
The updated Banff classification highlighted two major phenotypes of ABMR
-phenotype 1 C4D +VE ABMR early post transplantation period in highly sensitized patients
– Phenotype 2 C4D -ve ABMR, late post-transplant, due to denovo anti HLA class11 antibodies and associated with late graft loss but again can occur acutely in highly sensitized patients with persistent DSA despite desensitization, complement independent direct endothelial damage by antibody dependent cell mediated cytotoxicity including NK, neutrophils.
In this cohort group the incidence of ABMR is higher than TCR with C4D -ve ABMR only 18% while C4D +VE predominates of total 72%, this can be explained by higher rate of presensitization with blood transfusion, pregnancies, nonadherence
The prevalence C4D negative ABMR reported similar rate or even higher in some studies as high as 49% (34,33).
Although C4D -ve ABMR shows better graft survival rate in this study but its overall associated with progressive graft loss over time.
Limitations:
Retrospective design
No biopsies for the C4D negative ABMR on FU
Quantitative DSA not available
Type of the study
retrospective cohort study with FU outcome
level of evidence :
2B
Appropriate management of renal allograft dysfunction is important for long-term graft survival, Biopsy is still the gold standard for diagnosis of the cause of early and late graft injury.
Acute antibody mediated rejection (AMR) is responsible for 20-48% of acute rejection episodes and has worse prognosis than acute cellular rejection (ACR)
AMR is caused by alloantibodies against donor specific HLA molecules or other cell antigens, they attack peritubular and glomerular capillaries.
C4d is a fragment of complement C4b, an activation product of the classic complement pathway, it binds covalently to endothelial cell surface and extracellular matrix component making a stable molecule easily detected by immunohistochemistry.
Detection of C4d is considered “a footprint” of an antibody interaction and is the best single marker of complement fixing circulating antibodies.
AMR presents with 2 major phenotypes:
first type appears early post transplant in pre-sensitized patients and more likely to be C4d positive
second type appears late post transplant due to de novo DSA and is likely to be C4d negative and may occur acutely in highly sensitized patients with persistent DSA.
C4d staining is a tool for diagnosis of antibody mediated graft injury either early or late post transplant.
C4d negative may be due to technical issues, poor complement fixation by some DSAs, complement independent pathway of AMR, Autoantibodies to angiotensin II type 1 receptor are associated with fibrinoid necrosis resembling AMR but with absent C4d deposition.
Studies showed that C4d negative AMR tend to occur late post transplant with subclinical presentation with better graft outcome than C4d positive AMR
Materials and methods:
Retrospective study aimed to determine the incidence and clinical outcome of C4d negative AMR
It included indicated diagnostic renal allograft biopsies that revealed immunological rejection and biopsies from patients with ABO incompatible transplants.
Biopsies were processed for light microscopy and C4d immunohistochemistry and were classified according to Revised Banff’ 13 diagnostic categories.
Study design:
The biopsies were categorized into 3 groups, group1 with biopsies revealing AMR, group2 with combined ACR and AMR and group 3 with ACR.
group1 and group2 were subdivided according to absence or presence of C4d deposition.
All patients with rejection received therapy according to type of rejection.
Patient death or dialysis dependency was the proposed endpoints of the study.
In this study, the incidence of C4d negative AMR was 18.2% while C4d positive was 72.3%
C4d positive and C4d negative AMR have similar frequency when concurrent cell mediated rejection is present and both may occur early or late post transplant.
C4d negative AMR was associated with better graft survival but with poorer graft function.
C4d negative AMR needs to be treated aggressively as C4d positive AMR irrespective of concurrence or absence of TCR
Prospective studies are needed to determine long term outcome of C4d negative AMR.
Limitations:
C4d negative AMR wasn’t re-biopsied during follow up so couldn’t determine possibility of conversion of C4d negative to C4d positive.
Quantitative DSA estimation wasn’t done
It couldn’t determine risk factors for development of C4d negative
type of study
Retrospective study
level of evidence: 3
Various immunological and non-immunological causes of renal allograft dysfunction (RAD) have been identified, of which humoral (antibody/B-cell mediated) and cellular (T-cell mediated)are the most common. Alloantibodies preferentially attack the peritubular and glomerular capillaries and T- cells infiltrate tubules and arterial endothelium-also both have different treatment options. ABMR have antibodies which can be due novo or preformed and directed against both HLA and non-HLA molecules.C4d deposition is associated with circulating antibody to donor HLA Class I/II antigens and is specific for complement fixing circulating antibodies to the endothelium.
A retrospective study conducted from January 2013 to December 2015,in which 934 patients with graft biopsies revealing immunological rejection were being included. In this study, biopsies were classified according to Revised Banff’13 diagnostic criteria’s. All the biopsies were compared for mean post transplant and mean follow-up time period in months. . S Creatinine levels (mg/DL) at the time of biopsy and at last follow-up, patient and graft survival were being compared. The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%, of which C4d-negative rejections were 18.48% and 18.7% in Group 1 (pure ABMR)and Group 2(combined ABMR with concurrent TCR), respectively. The mean Serum Cr at the end of three years follow-up in patients with C4d-negative rejections were comparatively higher. C4d-positive and C4d-negative ABMR shows similar frequencies of concurrent cell- mediated rejection and both can occur early or late post transplant. Graft outcomes were comparatively better in patients who developed C4d-negative ABMR as compared to C4d-positive positive ABMR.
Two major phenotypes of ABMR highlighted at the meeting :
1- Appears early in the post transplant period in a pre sensitized patient and is more likely to be C4d positive.
2- Develops late post transplant, is due to de novo DSA development, and is likely to be C4d negative. This type is most important, as it is responsible for late graft los
C4d negative result may be due to technical errors , different method of C4d detection ,poor complement fixation by DSA , complement independent pathway ABMR , antibodies against And II type 1 receptors , NK Fc receptors may have a role in acute rejection with C4d negative staining.
Limitations of the study ;
1-No re-biopsy during follow-up.
2- No quantitative DSA estimation was done.
Thanks Batool
Renal allograft dysfunction is
common and critical for long term graft function and survival. So we need accurate diagnosis for appropriate management .
Various causes of immunological and nonimmunological of renal graft dysfunction have been identified.
And there are several types of rejection that can occur in a RA including hyperacute, accelerated acute, acute, and chronic active rejections.
The immune system has the capability to recognise the transplanted graft as foreign and mediate an immune response through complex immunological pathways, its divided into cellular and humoral pathways.
For diagnosis biopsy remains the gold standard and provides valuable insights
into the pathogenesis of early and late allograft injury.
Acute ABMR is seen in 5-7% of transplant recipients and makes up for 20-48% of all acute rejections. Antibodies in AMR act on peritubular and glomerular capillaries, while the T cells act on tubules and arterial endothelium in T cell mediated rejection.
Retrospective study was been conducted in 987 subjects to determine the incidence and outcome of C4d-negative ABMR. Biopsies were categorized through light microscopy and C4d immunohistochemistry into Group 1: pure ABMR, Group 2: combined ABMR with concurrent T-cell-mediated rejection (TCR), and Group 3: pure TCR. Groups 1 and 2 were further subgrouped as C4d positive (Group 1a and 2a) or C4d negative (Group 1b and 2b).
• Immunological injury was confirmed in 43.3% of these biopsies
• the most common form rejection was mixed rejection (60.6%), followed by pure ABMR (27.7%) and lastly pure TCMR (11.3%).
• C4d positive ABMR is more common (around 72%) thanC4d negative ABMR (around 18%)
• Patients with ABMR who are C4d positive usually present earlier(C4d negative ABMR tends to occur later
• Patients with ABMR who are C4d positive has lower graft survival (77.7%). than those who are C4d negative (90.2%), this means that it is a marker for severity and has a prognostic impact.
• C4d negative patients have a higher rate of developing transplant glomerulopathy if untreated
• Serum creatinine is found to be higher in C4d negative ABMR when compared to positive ones and this may be explained by the non-aggressive treatment of C4d negative ABMR
• This shows that C4d-negative injury does affect the graft function if not treated aggressively and causes graft attrition in the long-term .
All the patients who developed rejection
were subjected to antirejection regimen which was decided according to the type of rejection reported on biopsies. Patients who developed cellular rejection were treated with intravenous methyl prednisolone 500 mg/day for
3–5 days; rabbit anti-thymoglobulin (r-ATG) 1.5 mg/kg BW was added for resistant cases.
C4d-positive ABMR was treated with three sessions of plasmapheresis (with 40 mL/kg BW/session) followed by i.v. Ig 5 g on alternate days and subsequent bortezomib, 1.3 mg/m2 on days 1, 4, 7. and 11 or rituximab, 50–300 mg single dose.
For combined TCR + ABMR, methylprednisolone and rATG (in
doses mentioned above) were administered followed by plasmapheresis.
Patients with C4d-negative rejection were offered monotherapy with either bortezomib or rituximab. None of these patients underwent therapeutic plasma exchange.
The overall incidence of ABMR was higher this could be attributed to various causes such as transplantation across HLA barriers, multiple transfusions, presensitized patients, and multiple pregnancies in women apart from low hygienic conditions prevailing
no difference in demographic data of patients.
What is the kind of this study?
a retrospective cohort study
What is the level of evidence?
level III
Thank You
A retrospective study to determine the incidence and outcome of C4d-
negative ABMR.
introduction,
Study design,
a. Group 1: pure ABMR (27.7%)
b. Group 2: combined ABMR with concurrent TCMR (60.6%)
c. Group pure TCR (11.3%).
Groups 1 & 2 further divided into:
Anti rejection regimen used,
Results
What is the kind of this study?
Retrospective study
What is the level of evidence?
level of evidence 3
Well done
Please summarise this article :
1- technical issues related to the type of the fixative of complement.
2- different methods of C4d detection.
3- poor complement fixation by some DSA.
4- complement-independent pathways of AMR.
5- auto-antibodies to Angiotensin II type 1 receptor. they can mediate graft loss and fibrinoid necrosis similar to rejection but with -ve C4d.
6- Fc receptors on NK cells may play a role in acute rejection with -ve C4d.
7- Loupy et al., reported significant proportion of patients with AMR transit between -ve C4d staining and +ve C4d deposition.
1- incidence of C4d -ve AMR is strickingly low. it is of 18.2 % in patients with AMR
2- clinical symptoms are not helpful in determining risk factors for C4d -ve AMR.
3- graft outcome was better in those with C4d -ve AMR. however, longitudinal studies are required to know the significance and long-term outcome.
4- molecular studies are helpful to determine antibodies responsible for C4d-ve AMR to design targeted therapies.
What is the kind of this study?
What is the level of evidence?
Well done
C4d negative Antibody mediated rejection: Pathologist’s Perspective and clinical outcome
This is retrospective study if indicated diagnostic renal allograft biopsies performed from July 2013 to December 2015. Patient whose biopsies demonstrated immunological rejection were induced but those who were ABOI and lost follow ups are excluded.
Study design
G1- pure ABMR , G1a- C4d positive, G1b- C4d negative
G2- combined TCR and ABMR, G2a- C4d positive and G2b- C4d negative
G3- pure TCMR
Outcome:
primary end point -Death censored graft loss
Secondary end point – patient death or dialysis dependency
Results
404 patients included and 80.4% Males.
Mean posttransplant period
Time: 5 days to 8 years
C4d + ABMR presented at 1.32 SD 0.28 months,
C4d -ABMR -TCR presented at 1.33 SD 0.15 months
C4d-ABMR- 1.51 SD 0.27 months
TCR pure- 1.61 SD 0.66 months
C4d -ABMR was 20.2% within one year
75.5% C4d + ABMR
When C4d- alone AR apperas later than C4d + ABMR but when it combined with TCR it present earlier 1.32 vs 1.51 months
C4d + ABMR when combined with TCMR – appears late than 1.98 months
Prebiopsy SCr
Highest in C4d + ABMR (2.5 SD0.24)
C4d-ABMR- 2.11 SD 0.32, but P value not significant
Subgroup noted pure C4d + ABMR had higher Scr level 2.74 SD 0.35
HLA mismatch
Better matched Ts had better graft survival
Current Scr
Mean Scr in C4d – was 1.85 SD 0.56 while Cd4+ was 1.93 SD 0.21
Mean SCr pure ABMR with C4d – was highest
Graft survival
Overal graft survival in C4d- was better
6.4% in C4d + ABMR had graft loss
TCR + C4d+ had 4.6% Graft loss
C4d-ABMR had 2.5%
C4d+ vs C4d- 5.2% vs 2.5%
In conclusion, C4d negative ABMR and C4d positive had similar frequencies of concurrent TCMR which can occur early or late.Graft outcome were comparatively better in C4d – cohort.
it is a retrospective study with level of evidences 3
Well done
Renal allograft dysfunction is a common and complex problem of Renal Tranx, several types of rejection that can occur in allograft including hyperacute, accelerated acute, acute, and chronic active rejections.
In spite of significant developments in the diagnostic methodologies for
diagnosis of allograft dysfuncation, biopsy still remains the gold standard and provides valuable insights into the pathogenesis of early and late allograft injury.
Alloantibodies preferentially attack a different “location,” namely the peritubular and glomerular capillaries in contrast to T- cells which characteristically infiltrate tubules and arterial endothelium. ABMR is caused by antibodies directed against donor-specific human leukocyte antigen (HLA) molecules or other cell antigens. The most common
mechanism underlying ABMR is an antibody response that originates from previous antigenic exposure or de novo development of DSA.
Feucht et al showed that peritubular capillary (PTC) C4d deposition in RT biopsies is strongly associated with a poor prognosis and raises the
possibility that antibodies are responsible for graft loss.
C4d is a fragment of complement C4b, an activation product of the classic complement pathway.
antibodies can lead to graft accommodation through upregulation of complement regulatory or cytoprotec-tive proteins. It depends on the specificity and concentration of the antibodies: high-titer DSAs may cause ABMR, while recipients with low-titer DSAs can develop accommo-
dation. Endothelial cells are targets for immunemediated assaults through antiendothelial cell antibodies.
Study Place pathology department, Civil hospital campus, India from Jan 2013 to end of 2015. Type : a retrospective study 987 patients who underwent renal allograft biopsies, all samples are subjected to LM and C4d IHC. S.cr at time of biopsy and follow up is obtained. Time post-transplantation ranges from 5 days to 8 years. Inclusion Criteria: patients whose biopsy showed evidence of rejection. Exclusion Criteria: ABO-incompatible transplant, lost to follow up.
Results:
43% of the biopsies revealed an immunological injury, 27% of them showed pure ABMR, 60% combined TCMR+ABMR,11% pure TCMR, 18% of ABMR were C4d negative ,Mean Scr at the C4d negative ABMR was higher as compared to C4d positive after 3 years of follow up, Overall graft survival was better in C4d negative patients.
conclusion:
The incidence of C4d negative ABMR is low in this study.
Graft outcomes were better in C4d negative ABMR patients.
It is aretrospective study level 3.
Well done
I. C4d-Negative Antibody-Mediated Rejection: A Pathologist’s Perspective and Clinical Outcome
Please summarise this article
This study retrospectively looked into the incidence & outcome of C4d-negative ABMR.
A total of 987 renal allograft biopsies were studied using LM & C4d IHC on paraffin sections & reported according to modified Banff’s criteria.
Patients who underwent ABO-i transplants & those lost for FU were excluded (5.3%).
Of the remaining 934 allograft biopsies, 43.3% (404) revealed immunological injury on histology & were categorized as:
1. Group 1: pure ABMR (27.7%)
2. Group 2: combined ABMR with concurrent TCMR (60.6%)
3. Group pure TCR (11.3%).
Groups 1 & 2 further divided into:
– C4d positive (Group 1a & 2a)
– C4d negative (Group 1b & 2b).
Overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27% (C4d-negative rejections were 18.48%
in Group 1 & 18.7% in Group 2).
The incidence of C4d-negative ABMR was less than that of C4d-positive ABMR.
Mean S.Cr at the end of 3 years FU in patients with C4d-ve rejections was comparatively higher.
Anti-rejection regimen was given to all who developed rejection:
– ACR: IV methylprednisolone 500 mg/day for 3–5 days; r-ATG 1.5 mg/kg BW added for resistant cases.
– C4d-positive ABMR: 3 sessions of PP 40 mL/kg BW/ session followed by IV Ig 5 g DAD & subsequent bortezomib, 1.3 mg/m2 on days 1, 4, 7. & 11 or rituximab, 50–300 mg single dose.
– Combined TCR + ABMR: methylprednisolone & rATG were given followed by PP.
– C4d-negative rejection: only monotherapy (either bortezomib or rituximab).
Outcome
The mean S. Cr level in C4d-ve ABMR was higher compared to other groups.
C4d+ve group was treated aggressively with PP compared to the C4d-ve group, so the SCr levels were less than in patients with C4d-ve ABMR.
This shows that C4d-ve injury does affect the graft function if not treated aggressively & causes long-term graft loss.
Graft survival was better in the C4d-ve group despite the higher mean S. Cr levels in this group (Fig 1 & 2).
The incidence of C4d-ve ABMR was low & the demographic or clinical parameters were unhelpful in
determining risk factors of development of C4d-ve ABMR.
C4d+ve & C4d-ve ABMR show similar frequencies of concurrent CMR & both can occur early or late post-transplant.
Although graft outcomes were better in patients with C4d-ve ABMR as compared to C4d+ve ABMR, however longitudinal prospective studies are needed to determine the clinical significance & long-term outcome of C4d-ve ABMR.
Molecular studies might helpful to determine the responsible antibodies to guide treatment approach in
this group & thus improve graft survival these patients.
Definitions:
– C4d-positive ABMR: presence DSA+ glomerulitis &
PTCs on histopathology + PTC C4d deposition with an intensity of ≥2+ on IF & C4d >0 by IHC.
– Acute TMA) in the absence of any other cause was also reported as ABMR.
– Glomerulitis (g>0) – defined as the % of glomeruli with leukocytes infiltration in the capillaries(Score g1, g2, & g3 if <25%, 26%–50%, & >50% of glomeruli affected).
– Peritubular capillaritis – % of PTCs in the cortex that contained neutrophils or mononuclear cells (ptc1: <10% PTCs revealed cells, ptc2:10% of PTCs in-filtrated by <5 cells, ptc3:10% of PTCs revealed >10 cells/PTC).
– C4d deposition scoring by IHC– positive if grade was >C4d0 (C4d1, C4d2, & C4d3 if 1–9%, 10%–50%, & >50% PTCs affected, respectively).
– C4d-negative ABMR – biopsies with C4d <2 on IF or C4d < 0 on IHC, ptc> 0 & g >0, g+ptc ≥2, or g>0 or ptc >0 & acute TMA in the absence of other causes.
– TCR – biopsies with tubulitis (t>1) along with vasculitis (v>0) &/or interstitial inflammation (i>2).
– ABMR with TCR – Biopsies with C4d-ve ABMR+ histological evidence of TCR also had g ≥1 to qualify the criteria of ABMR.
– DSA (anti– HLA Class I & II antibody specificity) performed using SABs on Luminex. MFI >1000 was considered positive.
file:///C:/Users/TOSHIBA/AppData/Local/Temp/msohtmlclip1/01/clip_image002.gif
Figure 1. Kaplan–Meier survival plot depicting the renal allograft survival.
file:///C:/Users/TOSHIBA/AppData/Local/Temp/msohtmlclip1/01/clip_image004.gif
Figure 2. Kaplan–Meier survival plot depicting the patient survival.
What is the kind of this study?
Hospital-based retrospective cohort study
What is the level of evidence?
Level II
Level III?
Yes a retrospective level III Study
1)summary:
Renal transplantation is optimal treatment option for ESRD patients.
graft loss is a challenge facing renal transplantation which it could be multi-factorial such as immunological causes include:acute graft rejection(ABMR&TCR).
Renal allograft biopsy is the gold standard tool for diagnosis of RAD.
In all renal TX the ABMR occur in 5 to 7% and 20 -48% of AR episodes.
ABMR has worse prognosis and its modalities of therapy is different from TCR,and is caused by antibody direct against DSA donor.
C4d is a fragment of complement of c4b activation product of the classical complement pathway.
Feucht et al suggested that capillary c4 deposition was an evident for humeral alloreactivity against the graft.
capillary c4d staning is pathogonamic for antiHLA alloantibody and this antibody can be mediated through complement dependent and independent mechanism.
This study performed in Hospital Campus and Ahmedabad, Gujarat, India to determine the incidence of c4d negative ABMR and evaluate their outcome in term of graft function and survival.
This retrospective study was carried on from 2013 to Dec 2015 for 934 graft biopsy and the graft function was monitored by Scr level(mg/dl) the technique was used c4d IHC formalin-fixed biopsy specement to examine by light microscopy.
The study include all patients revealed immunological rejection in graft biopsy but exclude:
Baniff13 diagnostic classify for RA biopsy is used in this study
Optimal adequate biopsy at least 10 glomeruli +2 arteries
marginal biopsy 7-9 glomeruli +1 artery
minimal acceptable biopsy 7 glomeruli +1 artery
presence of DSA ,c4d +ve,glomerulitis and capillaritis all are used to diagnosed ABMR
They used IHC parrafin in embedded to detect c4d deposition in PTC graded based on intensity c4d +ve if more than 0 and IF 2 or more
Score of glomerulitis based on%infiltration of leukocytes
based on type of rejection ,all patients received specific anti rejection therapy and c4d negative patients received either bortezomib or rituximab
The end point of patients in the study is death or patients dialysis dependent
Scr in c4d negative group was higher than c4d +ve group since c4d +ve group received aggressive anti rejection therapy
Factors influence the c4d negative result:
Conclusion:
Incidence of c4d negative antibody mediated rejection
TCR has the same incidence of occurrence with c4d +ve and negative
Graft outcome better on c4d -ve than +ve
Requirement of prospective studies to know the clinical significance and long term outcome of c4d -ve ABMR
2) The type of study is retrospective study
3) level 3
Thanks
Results:
43% of the biopsies revealed an immunological injury
27% of them showed pure ABMR.
60% combined TCMR+ABMR.
11% pure TCMR.
18% of ABMR were C4d negative
Mean Scr at the C4d negative ABMR was higher as compared to C4d positive after 3 years of follow up.
Overall graft survival was better in C4d negative patients.
Limitations:
Conclusion:
The incidence of C4d negative ABMR is low in this study.
Graft outcomes were better in C4d negative ABMR patients.
Retrospective study
Level III
Thanks
This is the retrospective study of level III to determine the incidence and outcome of C4d-negative ABMR where the 987 renal allograft biopsies from 987 renal transplant recipients were studied.
All renal allograft biopsy samples were examined by light microscopy using standard stains and C4d immunohistochemistry on paraffin sections and reported according to modified Banff’s criteria. The renal graft biopsy results divided into groups.Group 1: pure ABMR, Group 2: combined ABMR with concurrent T-cell-mediated rejection (TCR), and Group 3: pure TCR. Groups 1 and 2 were further subdivided as C4d positive (Group 1a and 2a) or C4d negative (Group 1b and 2b). Graft function was measured by serum creatinine (SCr) level (mg/dL).
Inclusion criteria: Patients with biopsies revealed immunological rejection were included in the study.
Exclusion criteria: Patients who underwent ABO incompatible transplants and those who were lost for follow- up were excluded from the study.
-Adequacy of graft biopsy specimen defined as with at least 10 nonsclerotic glomeruli and two arteries. Specimens with less than 7 glomeruli or no artery considered inadequate biopsy for interpretation.
Definition of C4d-positive ABMR :was defined if there was the presence of circulating DSA along with glomerulitis and peritubular capillaritis on histopathology with the presence of diffuse C4d deposition across the PTC membranes with an intensity of ≥2+ on immunofluorescence and C4d >0 by immuno- histochemistry on paraffin sections.
Results:
The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%, of which C4d-negative rejections were 18.48% and 18.7% in Group 1 and Group 2, respectively. The mean SCr at the end of three years follow-up in patients with C4d-negative rejections was comparatively higher. C4d-negative ABMR, recently included in Banff’13, has a low incidence, usually presents early after transplantation but carries better outcome than C4d-positive ABMR.
Thanks
The most recent Banff meeting update high- lights two major phenotypes of ABMR. The first type appears early in the posttransplant period in a presensitized patient and is more likely to be C4d positive.The second type develops late posttransplant and is due to de novo DSA development and is likely to be C4d negative.
C4d is a fragment of complement C4b, an
activation product of the classic complement pathway.
Detection of C4d is
regarded as an indirect sign, a “footprint” of an antibody response.
C4d deposition is strongly associated with circulating antibody to donor HLA Class I/II antigens and is currently the best single marker of complement-fixing circulating antibodies to the endothelium.
Antibodies can mediate
endothelial injury through complement-depen-
dent and independent mechanisms.
On the other hand, under certain conditions, antibodies can lead to graft accommodation through upregulation of complement regulatory or cytoprotective proteins.
It depends on the specificity and
concentration of the antibodies: high-titer
DSAs may cause ABMR, while recipients
with low-titer DSAs can develop accommodation.
A total of 987 renal allograft (RA) biopsies obtained from 987 RA recipients were studied from January 2013 to January 2016. All samples were subjected to light microscopy using standard stains and C4d immunohistochemistry on paraffin sections and reported according to modified Banff’s criteria. Adequate biopsies with immunological injuries were categorized as Group 1: pure ABMR, Group 2: combined ABMR with concurrent T-cell-mediated rejection (TCR), and Group 3: pure TCR. Groups 1 and 2 were further subgrouped as C4d positive (Group 1a and 2a) or C4d negative (Group 1b and 2b). Graft function was measured by serum creatinine (SCr) level (mg/dL). Of the 987 biopsies, 43.3% (404) biopsies revealed immunological injury. Of these, 27.7% of the biopsies revealed pure ABMR (Group 1), 60.6% revealed combined ABMR with TCR (Group 2), and 11.3% revealed pure TCR (Group 3). The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%, of which C4d-negative rejections were 18.48% and 18.7% in Group 1 and Group 2, respectively. The mean SCr at the end of three years follow-up in patients with C4d-negative rejections was comparatively higher. C4d-negative ABMR, recently included in Banff’13, has a low incidence, usually presents early after transplantation but carries better outcome than C4d-positive ABMR.
The demographic profile or clinical symptoms are not helpful in determining the risk factors for the development of C4d-negative ABMR
Prebiopsy serum creatinine levels:
On comparing the serum creatinine levels of each subgroup, it was observed that patients with pure C4d positive had a higher SCr level (2.74 ± 0.35) than any other subgroup.
Qualitative donor-specific antibodies estimation:
100% positivity in patients having ABMR. DSA was absent in all the cases with TCR.
Human leukocyte antigen match:
the better the HLA match, better was the graft survival in all the groups.
Present serum creatinine levels:
Since the C4d– positive group was treated aggressively with plasmapheresis as compared to the C4d- negative group, the SCr levels tended to be less than in patients with C4d-negative ABMR. This shows that C4d-negative injury does affect the graft function if not treated aggressively and causes graft attrition in the long-term .
Graft survival:
graft loss appears to be higher in the C4d-positive group as compared to the C4d-negative group (5.2% vs. 2.5%).
C4d-negative ABMR usually occurs
>12 months after transplantation but can occur
acutely in highly sensitized patients with
persistent DSAs (even after desensitization).
causes of C4d negativity:
Technical issues related to type of fixative
used
different methods of C4d detection.
poor complement fixation by some DSAs, complement-independent pathway of ABMR
Autoantibodies
to the angiotensin II type 1 receptor have also
been associated with graft loss and fibrinoid
necrosis that resembles alloantibody-mediated
rejection except for the common absence of
PTC C4d.
Fc receptors on NK cells (FcRIIA)
may also play a role in AR, and it is possible
that some examples of AHR in biopsies that
lack C4d are due to this mechanism.
The overall incidence of C4d-negative ABMR in this study was 18.2%, which is much less than C4d-positive ABMR (72.3%). C4d–negative ABMR when occurs alone, usually presents later than C4d-positive ABMR (1.51 vs. 1.32 months); however, if concurrent TCR is also seen then it usually presents earlier than C4d-positive ABMR (1.33 vs. 1.98 months).
This was an Institutional Review Board approved retrospective study.
Level 5 narrative review
Level III as it is a retrospective study
C4 d negative antibody mediated rejection a pathologist perspective and clinical outcome
Please summarise this article
Renal graft dysfunction is major concern post kidney transplant and it can be categorised into 4 forms. This can be hyper acute , accelerated acute or chronic rejection. Rejection can be categorised into either cell mediated rejection or antibody mediated rejection and any form of rejection has poor outcome. In general the acute antibody mediated rejection is seen in about 5 to 7% of kidney transplant recipients and makes up about 20 to 48% of all acute rejections. In case of antibody mediated rejection antibodies act on Peri tubular capillaries while in case of cell mediated rejection it occurs on due to effect of T cells on endothelium
C 4d is a product of classical compliment cascade and it attaches to endothelial cell surface C4 d detection is a sign of antibody mediated rejection while there can be a group of patients who have AMR are with negative CD4
Material and methods: This was a retrospective study between January 2013 to December 2015 and the purpose of study was to define the incidents an outcome of C4d negative rejection. In this study a total of 987 renal allograft biopsies were done. The samples were processed under light microscope with standard stains and immunohistochemistry on paraffin sections and reported it according to modified Banffs criteria . Group One -was pure antibody mediated rejection Group 2- was combined antibody mediated rejection with concurrent TCR Group 3 -pure T cell mediated rejection. Group One and two were further subdivided into C4d positive and C4d negative .
Definition of Cd4 positive and C4d negative ABMR-
C4d positive- Positive DSA with glomerulitis and peritubular capilliritis. Positive C4d across PTC membrane . Thrombotic microangipathy with no other cause was labelled as positive for ABMR.
C4d negative- Negative C4d on IHC and acute TMA with no other cause
Results
60.0% had mixed rejection, Pure ABMR in 27.7 and 11.3 had TCMR. Incidence of TCMR was similar in both C4d positive and C4d negative cases. In ABMR, C4d positivity was seen in 82% and negativity in 28%. C4d positive ABMR had earlier presentation than C4d negative . Serum creatinine was better in C4d positive group like due to better treatment in this group. The study confirmed the prognostic value of C4d positivity . Graft survival was 77.7% in C4d positive group while it was 90.2 in negative group. Incidence of TG was high in C4d negative group.
What is this type of study.
A retrospective study
Level of evidence
level of evidence is -111
Thanks
C4d-Negative Antibody-Mediated Rejection: A Pathologist’s
Perspective and Clinical Outcome:
A retrospective study to determine the incidence and outcome of C4d-
negative ABMR.
A total of 987 renal allograft (RA) biopsies obtained from 987 RA
recipients were studied from January 2013 to January 2016. They
divided to groups:
Group 1: pure ABMR, 27.7%, C4d negative incidence 18.48%
Group 2: combined ABMR with concurrent T-cell-mediated rejection
(TCR), 60.6%, C4d negative incidence 18.7%.
Group 3: pure TCR, 11.3%.
Groups 1 and 2 were further sub grouped as C4d positive (Groups 1
and 2 were further sub grouped as C4d positive (Group 1a and 2a)
or C4d negative (Group 1b and 2b).
The overall incidence of ABMR (pure ABMR + ABMR with TCR) was
36.27%.
The mean SCr at the end of three years follow-up in patients with
C4d-negative rejections was comparatively higher.
C4d-negative ABMR, has a low incidence, presents early after
transplantation but carries better outcome than C4d-positive ABMR.
However, further long-term studies are still required for knowing the
clinical course over years.
Introduction:
Renal allograft dysfunction (RAD) is a common and complex problem
of RT.
Acute rejection (AR) episodes are a major determinant of RA survival
and long-term outcome of RT.
Acute antibody–mediated rejection (ABMR) occurs in 5%–7% of all
RTs and is responsible for 20%–48% of AR episodes.
The prevalence of chronic ABMR varies from 5% at one year to 20%
at five years.
De novo production of DSA against a transplanted donor organ can
lead to severe graft injury.
Detection of C4d is regarded as an indirect sign, a “footprint” of an
antibody response.
No functional role of C4d per se has been reported.
C4d deposition is strongly associated with circulating antibody to
donor HLA Class I/II antigens and is currently the best single marker
of complement fixing circulating antibodies to the endothelium.
C4d staining of RA biopsies has emerged as an important tool for the
diagnosis of antibody-dependent allograft injury independent from its
occurrence early or late after transplantation.
Graft survival:
The graft survival of all the groups is depicted in the Kaplan–Meier
survival plot. The overall graft survival appears to be better in the
C4d-negative group as compared to the C4d-positive ABMR group in
spite of the higher mean SCr levels in this group.
Discussion:
The most recent Banff meeting update highlights two major
phenotypes of ABMR. The first type appears early in the
posttransplant period in a presensitized patient and is more likely to
be C4d positive.
The second type develops late posttransplant and is due to de novo
DSA development and is likely to be C4d negative.
C4d-negative ABMR usually occurs >12 months after transplantation
but can occur acutely in highly sensitized patients with persistent
DSAs (even after desensitization).
Some causes of C4d negativity:
Technical issues .
Poor complement fixation by some DSAs.
Compliment-independent pathway of ABMR.
Autoantibodies to the angiotensin II type 1 receptor.
Fc receptors on NK cells (FcRIIA) may also play a role in AR.
Limitations:
Study cohort with C4d-negative ABMR was not re-biopsied during
follow-up. Hence, the possibility of C4d negative converting to C4d
positive that was demonstrated by could not be determined.
Level of evidence 111.
What is the kind of this study?
Retrospective study.
Thanks
C4d-Negative Antibody-Mediated Rejection: A Pathologist’s Perspective and Clinical Outcome
Various causes of immunological and nonimmuno logical renal allograft dysfunction (RAD) have been identified. There are several types of rejection that can occur in a RA including hyperacute, accele rated acute, acute, and chronic active rejections. The recognition of the transplanted organ as foreign is mediated by complex immunologic pathways, which in general terms can be divided into cellular (T-cell me- diated) and humoral (antibody/B-cell mediated) pathways.
Acute antibody–mediated rejection (ABMR) generally has a worse prog- nosis and requires a different form of therapy than T-cell-mediated rejection (TCR). Alloantibodies are now appreciated as important mediators of acute and chronic rejection, differing in pathogenesis or “nature,” from TCR. Alloantibodies preferentially attack a different “location,” namely the peritubular and glomerular capillaries in contrast to T- cells which characteristically infiltrate tubules and arterial endothelium. ABMR is caused by antibodies directed against donor-specific human leukocyte antigen (HLA) molecules or other cell antigens.
Showed that peritubular capillary (PTC) C4d deposition in RT biopsies is strongly associated with a poor prognosis and raises the possibility that antibodies are responsible for graft loss. C4d deposition is strongly associated with circulating antibody to donor HLA Class I/II antigens and is currently the best single marker of complemente fixing circulating antibodies to the endothelium. Capillary C4d deposition was an evidence for humoral alloreactivity against the graft. Demonstrated that staining of allograft biopsies for the fragment C4d is a specific and reliable method for identifying lesions due to allo-antibodies against HLA Class I and/or Class II antigens. The specificity of capillary C4d staining for anti-HLA alloantibody-depen- dent graft injury was further demonstrated recently by Böhmig. ABMR presents most of the time as severe allograft dysfunction and its prompt diagnosis and optimal treatment are essential.
This was an Institutional Review Board approved retrospective study of indicated diag- nostic RABs performed from January 2013 to December 2015. The demographic data regarding patient age, gender, indication for RA biopsy, and serum creatinine were collected. Graft function was measured by SCr levels (mg/dL). Formalin-fixed biopsy specimens were pro- cessed for light microscopy and C4d immuno- histochemistry as per manufacturer’s protocol. Membranous/lupus nephropathy slides were used as positive controls. Endothelial cells lining the medium caliber blood vessels were taken as internal control. Patients whose biopsies revealed immunological rejection were included in the study. Patients who underwent ABO incompatible transplants and those who were lost for follow- up were excluded from the study.
Histological categories were classified as per Revised Banff’13 diagnostic categories for RA biopsies. Definition of C4d-positive ABMR – ABMR was defined if there was the presence of circulating DSA along with glomerulitis and peritubular capillaritis on histopathology with the presence of C4d deposition across the PTC membranes. Others categories were defined based on the characteristics already mentioned: glomerulitis, peritubular capillaritis, C4d position. All the biopsies were compared for mean posttransplant and mean follow-up time period in months. SCr levels (mg/dL) at the time of biopsy and at last follow-up were compared. After analyzing the light microscopic features, the biopsies were categorized.
All the patients who developed rejection were subjected to antirejection regimen which was decided according to the type of rejection reported on biopsies. Patients who developed cellular rejection were treated with intra- venous methyl prednisolone 500 mg/day for 3–5 days; rabbit anti-thymoglobulin (r-ATG) 1.5 mg/kg BW was added for resistant cases. C4d-positive ABMR was treated with three sessions of plasmapheresis (with 40 mL/kg BW/session) followed by i.v. Ig 5 g on alter- nate days and subsequent bortezomib, 1.3 mg/m2 on days 1, 4, 7, and 11 or rituximab, 50–300 mg single dose. For combined TCR + ABMR, methylprednisolone and rATG (in doses mentioned above) were administered followed by plasmapheresis. Patients with C4d-negative rejection were offered monotherapy with either bortezomib or rituximab. None of these patients underwent therapeutic plasma exchange.
The most recent Banff meeting update high lights two major phenotypes of ABMR. The first type appears early in the posttransplant period in a presensitized patient and is more likely to be C4d positive. The second type develops late posttransplant and is due to de novo DSA development and is likely to be C4d negative. The second phenotype is an important factor in late graft loss; however, not many studies have been published regarding the clinical significance of this subset of patients. It appears that Class II HLA molecules may be responsible and that much of the endothelial damage is mediated by NK cells and, to a lesser extent, monocytes and neutro- phils; antibody-dependent cell-mediated cyto- oxicity. C4d-negative ABMR usually occurs >12 months after transplantation but can occur acutely in highly sensitized patients with persistent DSAs (even after desensitization).
The incidence of C4d-negative ABMR is strikingly low in our study. The demographic profile or clinical symptoms are not helpful in determining the risk factors for the development of C4d-negative ABMR. In our study, C4d-positive and C4d-negative ABMR shows similar frequencies of concurrent cell- mediated rejection and both can occur early or late posttransplant. Graft outcomes were com- paratively better in the cohort of patients who developed C4d-negative ABMR as compared to those who have C4d-positive ABMR. However, longitudinal prospective studies are required to know the clinical significance and long-term outcome of C4d-negative
This article is a primary study , in retrospective cohort.
Evidence is 3.
Well done Carols
C4d-Negative Antibody-Mediated Rejection: A Pathologist’s Perspective and Clinical Outcome.
It is a retrospective study to determine the incidence and outcome of C4d-negative ABMR.
Level of evidence III.
Introduction:
There are many immunological and non-immunological causes of graft dysfunction, one of the most important factor is rejection either TCMR or ABMR (the last one has a worse prognosis).
When antibodies directed against performed DSA or de novo DSA(HLA or non -HLA antigens) lead to activation of complement cascade and inflammatory cell infiltrates in PTC or glomeruli, and so on C4d stained and become a good marker of complement fixing circulating antibody to endothelium and ABMR.
Materials and Methods:
Retrospective study of indicated diagnostic RABs performed from January 2013 to December 2015.
Following serum .creatinine and kidney histological categories were classified as per Revised Banff’13 diagnostic categories for RA biopsies.
C4d- negative ABMR and evaluate their outcome in terms of graft function and survival. Patients of this group were compared to those having C4d-positive ABMR or TCR.
Study design:
According to biopsy result patients classified in to three groups
Group 1 :revealed ABMR.
Group 2 combined ACR +ABMR.
Group 3 with ACR.
And Group 1 and Group 2 were further subdivided on the basis of presence (C4d positive) and absence (C4d negative) of C4d deposits on PTC membranes.
Anti-rejection regimen:
Patients who developed cellular rejection were treated with intra- venous methyl prednisolone 500 mg/day for 3–5 days; rabbit anti-thymoglobulin (r-ATG) 1.5 mg/kg BW was added for resistant cases.
C4d-positive ABMR was treated with three sessions of plasmapheresis (with 40 mL/kg BW/session) followed by IVIg 5 g on alternate days and subsequent bortezomib, 1.3 mg/m2 on days 1, 4, 7. and 11 or rituximab, 50–300 mg single dose.
For combined TCR + ABMR, methylprednisolone and rATG were administered followed by plasmapheresis.
C4d-negative rejection were offered monotherapy with either bortezomib or rituximab without plasma exchange.
Results & Discussion:
-C4d-positive ABMR (Group 1) presented earlier than other groups and a higher S. creatinine level than any other subgroup.
-Follow up of creatinine reveled that C4d-negative ABMR injury does affect the graft function if not treated aggressively and causes graft attrition in the long-term.
-Graft survival: graft loss appears to be higher in the C4d-positive group as compared to the C4d-negative group.
Recent BANFF classification illustrated two ABMR phenotypes
The first one is mainly due to pre-sensitized recipient which is mainly C4d + and second one who developed de novo DSA which is likely is c4d negative ABMR which has effect on graft survival also.
Some studies have concluded that C4d- negative ABMR tends to occur slightly later post-transplant and has a subclinical presentation with better graft outcome than C4d- positive ABMR.
Graft survival of patients with C4d-negative ABMR was 97.5% as against C4d-positive ABMR which was 95.5% but also has poor graft function.
Conclusion:
C4d-positive and C4d-negative ABMR both can occur early or late post-kidney transplant.
Graft outcomes were better in patients who developed C4d-negative ABMR as compared to those who have C4d-positive ABMR.
Molecular studies would be helpful to deter- mine antibodies responsible for C4d-negative ABMR , trying to find targeted therapy to prevent the morbidity associated with this type of injury and improving graft survival .
Thanks
Banff 13 update consider C4d AMR as a separate intity for allograft dysfunction without a thorough elaboration on clinical and prognostic aspect of the condition.
This is retrospective study conducted in India between 2013 and 2016,
Aim : To determine the incidence and outcome of C4d negative AMR.
Study design:
987 patients presented with renal allograft dysfunction, had allograft biopsy .
categorized into 3 groups :
Group 1 AMR
Group 2 AMR and CMR
Group 3 CMR.
IHC for C4d was performedfor all patients with AMR and mixed AMR and CMR.
Follow up of 3 years post biopsy and treatment.
Results :
Revealed 36% with AMR pure and combined of which C4d negative was 18%.
C4d negative AMR Group showed higher serum creatinine at the end of 3 years of follow up.
C4d negative AMR is less common than C4d positive AMR. And it’s associated with better survival in comparison to C4d positive AMR.
Both can occur in combination with CMR.And both can happened early or late
C4d negative AMR is associated with DSAs against Class II HLA antigens ,they are mostly de novo anti HLA antibodies.
C4d negative AMR is associated with subclinical course and tend to occur slightly later and associated with better prognosis than C4d positive AMR.
the study suggested that a non complement fixing DSAs with antibody dependent cell=mediated cytotoxicity as the main mechanism of AMR.Similarly Autoan5ibodies to the angiotensin II type I receptor have also been associated with graft lossand fibrinoid necrosis resembling AMR but with no PTC C4d .
The study was retrospective study.
Level of evidence is 3
Thanks
This is retrospective study with level of evidence 3, performed between January 2013 to December 2015 in India. The histological diagnosis was independently reported by a panel of five pathologists and consensus generated was finally reported.
• Kidney biopsy specimens were processed for LM and C4d IHC. Patients whose biopsies revealed immunological rejection were included in the study.
• Patients who underwent ABO incompatible transplants were excluded from the study.
• Histological readings were classified as per Revised Banff’13 diagnostic categories.
• All the biopsies were compared for mean posttransplant and mean follow-up time period in months.
• Patient and graft loss data along with SCr levels were used to determine patient and graft survival.
The biopsies were categorized into three main groups.
• Group 1 pure ABMR
• Group 2 combined ACR +ABMR
• Group 3 pure ACR.
Alloantibodies attack peritubular and glomerular capillaries while T- cells infiltrate tubules and arterial endothelium.
Antibody mediated rejection in group 1 and 2 were further subdivided on the basis of positivity of C4d staining.
Results
404 out of 934 biopsies revealed immunological injury on histology.
• 27.7% biopsies revealed pure ABMR (Group 1)
• 60.6% mixed ABMR + TCR (Group 2)
• 11.3% pure TCR (Group 3).
The detection of C4d is not always indicative of antibody response and C4d negative does not exclude antibody mediated rejection .The incidence of C4d-negative ABMR was less than that of C4d-positive ABMR.
SCr level in C4d-negative ABMR (pure) was higher as compared to the control and other groups.
The overall graft survival appears to be better in the C4d-negative group as compared to the C4d-positive ABMR.
Graft loss appears to be higher in the C4d-positive group as compared to the C4d-negative group.
ABMR has a worse prognosis than T-cell-mediated rejection
The presence of peritubular capillary (PTC) C4d deposition in biopsies is associated with poorer prognosis.
Thanks
This article focus on C4d negative antibodies mediated rejection and it’s role in outcome of graft survival.
Kidney transplant remain the gold standard therapy in patients with chronic kidney disease.
Kidney allograft rejection is a common problem may lead to graft loss if not treated properly.
There’s Immunological and non immunological factors contribute to graft loss.
So several clinical presentations of allograft rejection hyperacute / accelerated acute / acute and active chronic.
there’s two different mechanisms of allograft rejection are cellular (T cell mediated rejection); Humoral ( B call mediated rejection).
B cell mediated rejection carry worse prognosis than T cell.
Alloantibodies are responsible for acute and chronic kidney transplant rejection.
The pathogenesis of allograft rejection is circulating DSA & de novo DSA which developed from exposure to previous antigen.
de novo DSA directed against MHC class I related chain A antigen and MHC class I related chain B antigen.
Another de novo DSA antibody are angiotensin type 1 receptor antigen and platelets specific antigen. renal biopsy remain the best diagnostic procedure in allograft rejection whereas the presence of C4d along peri tubular capillary and in endothelial cell of glomerulus carry poor prognosis for patients with allograft rejection.
C4d is fragment of C4b developed from activation of classic complement.
C4d is detected easily by immunohistochemistry and indicates for presence of DSA class I and class II antigens binding in endothelial tissue of graft leading to graft injury.
C4b depositions is specific markers and associated with ABMR.
C4d deposits in endothelial tissue for long time even after disappear of Ig and C1 ( it’s footprints along peritubular capillary).
Material and Method:
A retrospective cohort study done in Saudi from January 2013 to December 2015 based on diagnosis of renal allograft rejection on biopsy
Demographic data regarding age gender and indication of renal biopsy and graft function estimated by creatinine level.
renal biopsy done by using variety of staining. Formalin’s fixed biopsy were processed for light microscopy and C4d immunohistochemistry staining by use paraffin.
C4d staining negative biopsy restaining to avoid analytical errors.
Criteria including in this study are patients with immunological rejection and ABO incompatible.
Missing fallow up are excluded.
Reporting of renal allograft biopsy:
Adequate biopsy:
10 nonsclerotic glomeruli and 2 arteries.
Marginal biopsy:
7-9 glomeruli and one artery
Minimal acceptable biopsy
7 glomeruli and one artery
Inadequate biopsy
less than 7 glomeruli and no artery.
Histological categories are classified according to banff 13.
definition of C4d positive ABMR:
1. ABMR defined by presence of circulating DSA along with glomerulitis and peritubular capillarities on histology with C4d deposition across PTC membrane by immunohistochemistry and immunofluorescence.
2. Acute thrombotic microangiopathy whatever cause.
3. Glomerulitis with score g150%
– [ ] 4. Peritubular capillarities with neutrophils and mononuclear cell infiltration
ptc1 10%
C4d positive score ( C4d1 1/9%, C4d2 10-50%, C4d > 50%.
C4d negative score ( C4d 0 by immunohistochemistry.
TCR definition presence of tubulitis >1%
Vasculitis >0
Interstitial inflammation >2.
Mixed ABMR + TCR with C4d negative / and with positive C4d.
estimation of DSA for screening of anti HLA specific IgG Ab by single antigen bead and Luminex platform and mean fluorescence intensity.
Study design:
All biopsy were compared for mean post transplant and mean fallow up time period in months.
biopsy are divided into three different groups.
gp 1: positive ABMR / gp2: ACR + ABMR / gp 3 ACR
gp 1&2 subdivided into negative C4d and positive C4d PTC membrane.
Anti rejection regime:
All patients with rejection treated by Anti rejection regime.
patients with cellular rejection treated with Iv methyl prednisone
r-ATG add to resistant cases.
C4d positive treat by plasma exchange plus Ig plus bortezomib or rituximab
patients with mixed ABMR & TCR treated by methyl prednisone plus plasma exchange plus r-ATG
patients with C4d negative treated by rituximab or bortezomib.
Results:
Total patients in this study are 987 all biopsied only 5.3% excluded and 43.3% shows immunological injury
27% pure ABMR
60% mixed ABMR with TCR
11.3% pure TCR
C4d negative ABMR was less than c4d positive ABMR patients.
Graft loss:
Mean post transplant period time of allograft biopsy from 5 days to 8 years post transplant .
when biopsy were grouped into those present within one year and those present after one year incidence of c4d positive rejection more than negative c4d
patients with c4d positive have higher serum creatinine levels than those negative c4d.
patients with c4d positive treated aggressive with plasma exchange.
Graft survival: it’s better patients with C4d negative
Graft loss appear higher in patients with C4d positive in comparison with C4d negative
Discussion:
The most cause of ABMR in early post transplant is due to C4d positive.
in late post transplant is due to de novo DSA
C4d negative usually appear after 12 months post transplant but may appear acutely in high sensitised patients.
Autoantibodies like Angitensin type 1&2 receptor are associated with graft loss and fibrinoid necrosis
Incidence of ABMR are high in this study may related to multiple transplant or pre sensitised patients and multiple pregnancies and low hygienic conditions.
TCR usually present early than c4d positive ABMR
finally patients with c4d positive ABMR irrespective with presence or absence of TCR should be treated aggressively by plasma exchange plus r-ATG and Ig.
This article cohort retrospective study
level 3
Q 1.Summary:
· Acute rejection (AR) represents immune mediated graft damage which is associated with worse graft outcome.
· AR may be either :
o AMR ; with histopathological damage mainly in peritubular and glomerular capillaries.
o TCMR: damage mainly by lymphocyte infiltration in tubules, interstitium and arteriolar intima.
· AMR has 2 types;
o Type 1= early post transplant= preformed DSA = mainly c4d+ve.
o Type 2= late post transplant= denovo DSA= mainly c4d -ve.
· No sufficient evidence about relation to c4d positivity to graft outcome, with conflicting results.
· Many studies showed c4d -ve AMR may have better prognosis than c4d +ve. However, C4d-ve AMR still carries a risk of poor long-term graft outcome compared to those without rejection.
· Banff 2013 included c4d -ve AMR.
· C4d meaning:
o It represents activation of complement cascade.
o It remains bound to endothelium at site of injury by covalent bonds, so remain detectable for longer period even after disappearance of antibodies.
o So it represents foot print of AMR.
o It is associated with presence of circulating DSA (detected by Luminex SAB).
· Methods of c4d detection:
o IF (more sensitive), but mostly requires fresh tissue in frozen section 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 However, also IF can be done nowadays on paraffin section by special technique from available blocks.
· General histopathological definitions for biopsy adequacy:
o Adequate = 10 non sclerotic glomeruli+ 2 arteries.
o Marginal = 9 glomeruli+ one artery.
o Minimally accepted= 7 glomeruli+ one artery.
o Unaccepted= 7 glomeruli+ no artery.
· Definitions of positivity of c4d in biopsy:
o Circulating DSA+ intensity ≥ 2 by IF or >0 by IHC.
o C4d +ve may be scored to (1=< 10% of ptc, 2 if between 10-50% and 3 if > 50 % of Ptc involved).
· C4d negative AMR may be due to :
1. Technical errors
2. Different method of c4d detection (IF vs IHC).
3. Very low amount of c4d (difficult detection).
4. Poor complement fixation by DSA
5. Complement independent pathway AMR or antibody dependent cell mediated cytotoxicity.
6. Antibodies against Angiotensin II type 1 receptors
7. fc receptors of Nk may have a role c4d negative AMR.
8. Increased expression of endothelial transcripts causing endothelium injury.
9. Sure pure TCMR will have -ve c4d.however, mixed rejection may be either c4d +ve or -ve.
· C4d +ve staining without AMR:
o Not every c4d +ve means AMR: (sometimes accommodation to antibodies occurs so no immunologically mediated damage occur).
o ABO incompatible transplantation will have +ve c4d.
o Immune mediated kidney disease as lupus nephritis or anti phospholipid syndrome (either recurrent in the graft or infrequently present in donor and not detected). However, mostly here the c4d deposits will be mesangial or glomerular (not in peritubular capillaries).
· C4d -ve AMR in the current study were treated by either rituximab or bortezomib ( no PEX).
· Further longitudinal studies are needed to characterize c4d -ve AMR in relation to prognosis and ideal treatment.
C4d-Negative Antibody-Mediated Rejection: A Pathologist’s Perspective and Clinical Outcome
Renal allograft dysfunction (RAD) can be caused by a variety of things.
Rejection being one of the common causes.
It can be T cell (TCR) or antibody mediated rejection (ABMR) and biopsy is the gold standard for diagnosis.
ABMR has a worse prognosis and requires different treatment to TCR.
Alloantibodies cause damage to glomerular and peritubular capillaries vs TCR which T cells infiltrate tubules and arterial endothelium.
Antibodies can be pre-formed or de novo.
Directed against HLA- antigens but can also be directed against non-HLA molecules like MHC Class I related chain A antigens (MICA-A), MHC Class I-related chain B antigens (MICA-B), platelet-specific antigens, renin–angiotensin pathway molecules.
C4d deposition in peritubular capillaries is associated with presence of DSA and caries a poor prognosis.
C4d is a fragment of complement C4b a product of the classic complement pathway.
C4d remains bound to tissues for several days. It is a footprint of antibody response.
Antibodies cause allograft injury through complement activation.
May also upregulate complement regulatory protection and cytoprotective proteins leading to accommodation.
In this study renal allograft biopsies were reviewed to determine the incidence of ABMR and determine outcomes in terms of graft function and survival.
A retrospective study of renal allograft biopsies between m January 2013 to December 2015
Biopsies consistent with immunological rejection were included in the study
ABO incompatible transplants and patients lost for follow- up were excluded from the study
C4d positive ABMR was defined by presence of glomerulitis and peritubular capillaritis and C4d + staining in peritubular capillaries.
C4d negative ABMR was defined as above except for C4d <2 on immunofluorescence.
TCR- defined as tubulitis t>1, along with vasculitis (v>0) and/or interstitial inflammation (i>2).
DSA was determined by single antigen bead testing.
987 biopsies analysed. 5.3% excluded. Remained 934 biopsies.
404/934 (43.3%) had evidence of immunological injury.
27.7% biopsies revealed pure ABMR (group1)
60.6% mixed ABMR + TCR (group 2)
11.3% pure TCR ( group 3)
C4d-negative rejection was 18.48% in Group 1 and 18.7% in Group 2
Most of the rejections occurred in males 80.4%.
Times of biopsy ranged from 5 day to 8 years post-transplantation.
C4d+ ABMR presented earlier 1.32 ± 0.28 months
Followed by mixed ABMR+TCR
There were C4d+ than C4d – in the study.
C4d+ ABMR were more likely to have high serum creatinine than C4d -.
The mean HLA match was better in C4d+ patients (2.2) compared to C4d- (1.5)
C4d negative injury if untreated causes graft loss in the long run.
C4d negative had better graft survival compared to C4d+ ABMR
o graft loss reported in the C4d-negative ABMR
Conclusion
C4d negative causes graft injury if left untreated, however presence of C4d positive injury leads to more severe graft injury and loss of graft function.
A descriptive retrospective study
Level 3 evidence
I. C4d-Negative Antibody-Mediated Rejection: A Pathologist’s Perspective and Clinical Outcome
Graft dysfunction is a common scenario post- kidney transplant. Kidney rejection may be either hyperacute, accelerated acute, acute or chronic active rejection.
Rejection, which can be classified as either cellular or antibody mediated rejection (AMR), has major impact on graft prognosis. Acute AMR is seen in 5-7% of transplant recipients and makes up for 20-48% of all acute rejections. Antibodies in AMR act on peritubular and glomerular capillaries, while the T cells act on tubules and arterial endothelium in T cell mediated rejection.
C4d, formed by the classical complement pathway, gets attached to endothelial cell surface by sulfhydryl group. Although C4d detection is an indirect sign of antibody mediated injury, there is a group of AMR patients who are C4d negative.
This was a retrospective study (from January 2013 to December 2015) analysing the C4d negative AMR and comparing it with C4d positive AMR. The study included all graft biopsies done for indication showing rejection except those with ABO incompatible transplants and those who did not have adequate follow-up. Formalin-fixed kidney biopsies were assessed for light microscopic changes and C4d immunohistochemistry (IHC).
Standard definitions for adequacy of biopsy, parameters for glomerulitis, peritubular capillaritis, C4d scoring, C4d positive AMR, C4d negative AMR, T cell mediated rejection (TCR), and combined AMR with T cell mediated rejection were used.
Donor specific antibodies (DSA) were estimated using single antigen bead testing and normalized MFI >1000 was taken as positive.
C4d positive AMR: Circulating DSA with biopsy showing glomerulitis, peritubular capillaritis and C4d>0 on IHC.
C4d negative AMR: Circulating DSA with biopsy showing glomerulitis, peritubular capillaritis and C4d0 on IHC.
TCR: Biopsy with tubulitis (t>1), vasculitis (v>0) and interstitial inflammation (i≥2).
AMR with TCR: Combination of TCR and g≥1.
All the cases were divided into 3 groups. Group 1 included AMR, further divided into1a (C4d positive) and 1b (C4d negative).Group 2 included combined MAR and TCR, further divided into 2a (C4d positive) and 2b (C4d negative).Group 3 included TCR.
Patients with TCR were treated with methylprednisolone, and ATG if resistant.Patients with C4d positive AMR were treated with Plasmapheresis, IVIG, bortezomib or rituximab. For C4d negative patients, patients were given either rituximab or bortezomib without plasmapheresis. Combined AMR and TCR was treated with methylprednisolone and ATG.
Demographic profile, serum creatinine, HLA matching and graft survival was analysed.
Out of 987 biopsies, 404 (43.3%) had rejection, majority of whom were males. 27.7% of them had pure AMR, 60.6% had combined MAR and TCR while 11.3% had TCR alone. C4d negative AMR was less common with approximately 18% in both group 1 and group 2.
Mean serum creatinine at time of biopsy was higher in C4d positive AMR. DSA was present in all patients with AMR. Mean HLA match in C4d negative group was lower than in the C4d positive group. Serum creatinine levels on follow-up were highest in C4d negative AMR group, probably due to the type of treatment given in this group.
Graft survival was better in C4d negative group with higher graft loss in C4d positive group.
AMR has 2 major phenotypes: Phenotype 1 is seen early, in presensitized patients and C4d positive while phenotype 2 is seen later, is due to de novo DSA and likely C4d negative, due to class II HLA antibodies, causing late graft loss.
C4d negative AMR is usually seen more than 1-year post-transplant, but can be seen early in highly sensitized patients, has subclinical presentation with better prognosis than C4d positive AMR. C4d negative AMR, in association with TCR, can present earlier than C4d positive AMR.
The limitation of the study included lack of re-biopsy in C4d negative AMR to look for any change in C4d stain status, as a different group had shown that C4d negative AMR can become C4d positive.
The study concluded that C4d negative AMR incidence is low, with better graft survival but poorer graft function as compared to C4d positive AMR.
It is a retrospective cohort study
Level of evidence is Level 3