Anti-HLA antibodies is important for successful transplantation, and activation of the complement cascade is involved in antibody-mediated rejection.The analysis enrolled 1016 patients. The graft survival was 54% in patient with mplement-binding
donor-specific anti-HLA antibodies of as compared with patients with non–complement-binding donor-specific anti-HLA antibodies (93%) and patients without donor-specific anti- HLA antibodies (94%). The presence of comple-
ment-binding donor-specific anti-HLA antibodies after transplantation was associated with high a risk of graft loss . AMR , sever microvascular inflammation and C4D deposition is more and extensive in complement binding antibodies .
C1q assay may lead to distinguishing patients at risk, Although C4d is specific ,it is less sensitive as an indicator of antibodies rejection. The presence of complement-binding anti-HLA donor specific antibodies after transplantation is strongly associated with graft injury and loss
· What is the level of evidence demonstrated by this study? Level 11
· What this type of this study?Cohort prospective study
.
Wee Leng Gan
2 years ago
Complement-Binding Anti-HLA Antibodies and Kidney-Allograft Survival
This is a prospective cohort study with level II evidence involving kidney recipients at two transplantation centers in Paris between January 1, 2005, and January 1, 2011. The objective is to assess the association of complement-Binding Anti-HLA Antibodies and Kidney-Allograft Survival. A total of 1016 kidney transplant recipients recruited of which 700 patients without circulating donor-specific anti-HLA antibodies, 239 patients with non–complement-binding donor-specific anti-HLA antibodies, and 77 patients with complement-binding donor-specific anti-HLA antibodies.
Following renal transplant in the first year, a total 171 kidney transplant recipients developed acute rejection of which 96 patients; 56% had TCMR and 75 patients; 44% had AMR. TCMR is relatively higher among renal transplant recipients with donor specific anti HLA antibodies and C1q binding capacity. The median follow-up duration was 4.8 years. Those with donor specific anti HLA antibodies had poor renal graft survival especially those with C1q binding capacity donor specific DSA had poor 5-year graft survival.
The independent predictors of graft loss include low estimated GFR at 1-year, interstitial fibrosis and tubular atrophy, glomerular and peritubular inflammation, transplant glomerulopathy and the
presence of complement-binding donor specific anti-HLA antibodies.
In conclusion, complement-binding capacity of donor-specific anti-HLA antibodies allow early recognition of patients at high risk of allograft rejection and prompt action to be taken.
Mohamed Fouad
2 years ago
Complement-Binding Anti-HLA Antibodies and Kidney-Allograft Survival
Since the presence ofAnti-HLA antibodies is one of the main barriers for successful transplantation, and activation of the complement cascade is involved in antibody-mediated rejection. The aim of this paper to assess whether the complement-binding capacity of anti-HLA antibodies plays a role in kidney-allograft failure.
-Study type:prospective cohort study with level II evidence.
Methods:
Study included patients received kidney allografts in 2 centres in Paris between January 1, 2005, and January 1, 2011, in a population-based study. Patients were screened for the presence of circulating DSA antibodies and their complement-binding capacity. Rejection phenotype and the time to kidney allograft loss were assessed.
Results:
The primary analysis included 1016 patients. Patients with complement-binding donor-specific anti-HLA antibodies after transplantation had the lowest 5-year rate of graft survival (54%), as compared with patients with non–complement-binding donor-specific anti-HLA antibodies (93%) and patients without donor-specific anti HLA antibodies (94%). The presence of complement binding donor-specific anti-HLA antibodies after transplantation was associated with a risk of graft loss that was more than quadrupled when adjusted for clinical, functional, histologic, and immunologic factors. These antibodies were also associated with an
increased rate of antibody-mediated rejection, a more severe graft injury phenotype with more extensive microvascular inflammation, and increased deposition of C4d within graft capillaries. Adding complement-binding donor specific anti-HLA antibodies to a traditional risk model improved the stratification of patients at risk for graft failure.
There are therapeutic implications of knowing the pathways responsible for kidney allograft loss. Since promising therapeutic agents targeting complement (e.g., a C5 inhibitor [eculizumab] or a C1 inhibitor) are increasingly used in patients undergoing transplantation and ABMR.
One limitation of this study was that it was not designed to provide kinetics of the capacity of anti-HLA antibodies to bind complement
or the effect of treatment on these antibodies.
Nandita Sugumar
2 years ago
Summary
Anti-HLA antibodies can impact transplant outcome. One of the methods through which they act is by binding to complement, activating the complement cascade.
Patients with complement binding donor specific anti HLA antibodies appear to have very low rates of graft survival in comparison with recipients who have anti HLA antibodies that are not complement binding. Graft loss risk is more than 4 times. Other risk associations include AMR, and increased deposition of complement fraction C4d in graft capillaries.
Thus, complement binding anti HLA antibody assessment can help in identifying recipients who are at high risk of graft loss. Both C1q and C4d assessment are to be done. Even if C4d is negative, C1q has to be done because C4d lacks sensitivity although it is specific.
In conclusion, this study finds that post transplant presence of complement binding anti HLA DSA is strongly correlated with high risk of graft injury and graft loss. It is recommended to use this fact for risk assessment. C1q assessment is a significant tool in this aspect.
Level of evidence
Level of evidence is 2.
Type of study
Prospective cohort study
Dawlat Belal
Admin
2 years ago
Dear all thankyou for participation but l noticed some new names apart from the regular active ones.
This is fine but l would like to see contributions in the scenarios.
Please be encouraged to take part no problem to make mistakes that is how we all learned.
Abdullah Raoof
3 years ago
early studies recognized that ant HLA Ab are destructive .
-Then recognition that complement system activation is a key component of ABMR .
– recognition of c4d deposition to be a footprint of ABMR .
-The capacity of anti-HLA antibodies to bind C1q, may determines the cytotoxic potential of these antibodies,
-Patients with C1q-binding DSA had a lower estimated (GFR) than did patients with non–C1q-binding DSA and patients without donor-specific anti-HLA antibodies .
– Patients with DSA had significantly worse graft survival than patients without DSA .
– patients with C1q binding DSA had the poorest graft survival after transplantation as compared with patients with non– C1q-binding DSA and patients without DSA .
-patients with C1q binding donor-specific anti-HLA antibodies after transplantation had the highest risk of graft loss .
– the presence of complement-binding DSA detected in the first year after transplantation was an independent predictor of kidney-allograft loss after trans plantation and significantly improved individual risk stratification for graft failure.
-Patients with complement-binding DSA had a graft injury phenotype characterized by microvascular inflamation and complement split-product C4d deposition.
– Although the MFI of DSA is widely used in clinical practice for risk stratification, THIS study
showed that C1q-binding DSA remained strongly associated with the risk of graft loss regardless of the MFI
of the antibodies.
– From a prognostic perspective, this study provide support for the finding that C1q and C4d do not provide equivalent predictive information. C1q testing may help identify patients at risk, despite C4d negativity.
– this study found that C4d, although specific, lacks sensitivity as an indicator of humoral rejection.
– the presence of complement-binding DSA after transplantation is strongly associated with graft injury and loss and that in corporation of this risk factor improves risk stratification for graft loss.
TYPE OF STUDY
prospective cohort study
LEVEL
level II
ahmed saleeh
3 years ago
descriptive prospective cohort study
Level of evidence: II
anti-HLA antibodies are lymphocytotoxic,13 activation of the complement cascade has been considered to be a key component of antibody-mediated allograft rejection, and C4d deposition in renal capillaries has been considered the footprint of antibody-mediated allograft damage.
Plus complement fraction C1q, the first step in activation of the classic complement cascade.
Complement-binding donor-specific anti-HLA antibodies remained independently associated with the risk of kidney-allograft loss after adjustment for the mean fluorescence intensity of donorspecific anti-HLA antibodies
C1q-binding donor-specific anti-HLA antibodies remained strongly associated with the risk of graft loss regardless of the mean fluorescence intensity of the antibodies
may have implications for other transplanted organs such as the heart, lungs, and small bowel,
C1q testing may help identify patients at risk, despite C4d negativity
C4d, although specific, lacks sensitivity as an indicator of humoral rejection
promising therapeutic agents targeting complement (e.g., a C5 inhibitor [eculizumab] or a C1 inhibitor)
What is the level of evidence demonstrated by this study?
What this type of this study?
prospective cohort study with level 11 a of evidence
NEJM with high impact factor 91.24 in 2022
Introduction:
Kidney transplant survival and outcome compromised overtime due to the risk of antibody mediated rejection; anti- HLA antibodies considered the crucial part in the classic complement pathway activation resulted in antigen -antibodies alloimmune response with C4split product deposition which is the the foot print of alloimmune response. the study aims to assess the role of HLA Abs complement activation and the renal transplant graft failure
Methodology:
Population based prospective cohort study from two transplant centers in Paris, with external validation from cohort from third center sharing the same local registry database 2, they included 1016 patients with anti-HLA abs positive kidney transplant from the period between -2005-2011 and follow up at 0 time, 6, 12 months then annual review, last follow up extended till April 2012 with final outcome of graft survival. All included cases with ABO compatible transplantation with negative crossmatch for both T and B cells with similar allocation criteria from the three centers, the baseline characteristics shows in table 1, Patients with previous sensitization with DSA +ve pre transplants have more C1q complement activation compared to those with DSA -ve pre transplantation. They divided them into three groups according to the presence of complement binding anti HLA Abs, non-HLA DSA, non-complement binding DSA
Acute rejection defined as acute clinical renal function deterioration with proteinuria and histological diagnosis of rejection by protocol biopsies at one year post transplantation, in majority 845 cases while another 171 cases underwent graft biopsy as per clinical indication with acute graft dysfunction all biopsies reviewed based on updated Banff diagnostic score 0-3 by three independent pathologists and use IHC staining for C4D detection.
All have similar immunosuppression therapy for treatment of AR
Results:
Patients with C1q binding DSA have higher rate of AMR 48% and more sever microvascular injury with intimal arteritis , tubular and interstitial inflammation score of >2 , Tg , and higher score for C4D staining associated with lower GFR upon 1 year FU with progressive graft dysfunction as compared with non-complement binding DSA .Median FU 4.8 Years ( range 0.2-7) after transplantion and median FU for C1q binding DSA was 3.9 years
Renal survival was worse In those with anti HLA DSA , 5 year graft survival was 88% and even more worse graft survival was reported in those with anti HLA C1 q binding DSA( 54%), and also associated with higher rate of graft failure .
A prospective cohort with level of evidence :II
Allo -antibodies are still a major obstacle against successful transplantation. Anti-HLA antibodies, play significant role in kidney allografts failure due to allo immune response. Capillary C4d deposition has been considered as clue of antibody-mediated allograft damage and C1q properties of donor-specific anti-HLA antibodies may be specifically associated with AMR.
In this study,1016 patients from 2 centers with similar immunosuppressive protocols and induction regimens in both centers. They included CDC T and B cell negative cross match patients with follow up for 6 years. SAB Luminex was tested on day 0 and after 1 year with results of 700 patients had no DSA. 239 patients had non complement binding DSA and 77 patients with complement binding DSA. Kidney biopsies were done 1 year later or when clinical rejection was suspected. They concluded that complement binding anti HLA DSA were associated with lowest graft survival at the end of 6 years (54%) in comparison with 93% of those associated with non complement binding DSA.
Results support for the finding that C1q and C4d do not have similar predictive significance.C1q assay may aid identifying patients at risk, in spite of C4d negativity. Although C4d is specific ,it has insufficient sensitivity as an indicator of humoral rejection. The presence of complement-binding anti-HLA donor specific antibodies after transplantation is strongly associated with graft injury and loss. Considering this risk factor leads to better risk stratification for graft loss.
Very good highlighting the stronger risk significance of C1q HLA than C4d.
Theepa Mariamutu
3 years ago
Please give a summary of this article
HLA antibody testing has undergone a sea-change in last 5 decades. From requirement of a negative crossmatch for transplant, we have reached a stage where transplants are being performed without a physical crossmatch. The journey of development of antibody testing is an interesting one.
The sensitivity of the original CDC crossmatch increased on using antihuman globulin. The sensitivity of antibody testing increased by use of flowcytometry and introduction of microparticle technology (FlowPRA and single antigen bead/ Luminex) further enhanced both sensitivity and specificity.
MFI values provided by the Luminex technology give a semiquantitative assessment of antibody levels, signifying a relative fluorescence of a single bead after binding to antibody. MFI values do not correlate with clinical outcomes.
The limitations of MFI include lack of clarity with respect to cut-off values; lack of standardization with respect to vendor, source of antigen and test reagents as well as interlaboratory differences. Various factors interfering with the assay include IgM HLA antibodies and complement split products giving rise to falsely low MFI, as well as denatured antigen giving rise to false positive results.
The power of pattern-recognition is important in antibody detection, represented by CREGs (cross-reactive groups) – multiple HLA antigens reacting with a single antibody, nowadays called epitopes, eplets etc. So, bead reactivity should be interpreted with
The avidity and degree of antibody binding to antigen depends on the conformation/ physical alteration of the antigen leading to variable MFI levels with same antibody, making it very difficult to interpret the results. hence eplets (discontinuous polymorphic regions) are important part of compatibility testing.
DSA detected by antigen binding in vitro might not bind to the antigen in vivo due to altered expression levels of the antigen, but might bind once the antigen expression increases as after an inflammatory stimulus. So, the MFI levels cannot be a marker of pathogenicity of the antibody.
SAB assays identifying complement binding antibodies did not conclusively prove their association with poor outcomes, especially in pre-transplant period. Post-transplant IgG3 antibodies have been shown to be associated with poor graft outcomes. IgG1 DSAs have been shown to have good graft outcomes. IgG may not fix complement in vivo due to presence of complement inhibitors like heparin sulphate and DAF, non-hexamerization of Fc fragment and insufficient IgG molecule binding.
Each case is different and center-specific multidisciplinary approaches for kidney transplantation is need of the day. HLA antibody detection and interpretation of its results needs to be done in light of the history and clinical parameters of the patient.
What is the level of evidence demonstrated by this study?
type 2 evidence study
What this type of this study?
Prospective cohort study
Fine but you missed the results and conclusion of value of C1q fixing antibodies in risk stratification
MOHAMED Elnafadi
3 years ago
Please give a summary of this article
Despite the world wide advance in renal transplantations autoantibodies remain the major cause for graft loss. anti-HLA antibodies, plays a key role in the failure of kidney allografts due to alloimmune response, C4d deposition in renal capillaries has been considered the foot print of antibody-mediated allograft damage also complement fraction C1q properties of donor-specific anti-HLA antibodies may be specifically related to antibody mediated rejection.
Study summary
1016 patients from 2 centers were included in the study at 2005 2011 . Immunosuppressive protocols and induction regimens were same across both the centers. They included all CDC T and B cell negative cross match patients and followed them up for 6 years. The SAB Luminex was tested on day 0 and after 1 year. 700 patients had no DSA. 239 patients had non complement binding DSA and 77 patients with complement binding DSA. Kidney biopsies were done 1 year later or whenever the clinical nature of rejection was suspected. They concluded that complement binding anti HLA DSA were associated with lowest graft survival at the end of 6 years (54%) as compared to 93% of those associated with non complement binding DSA.
Coclusion
All results provide support for the finding that C1q and C4d do not provide equivalent predictive information. C1q testing may help identify patients at risk, despite C4d negativity, even C4d is specific,it lacks the sensitivity as an indicator of humoral rejection. The presence of complement-binding anti-HLA donor specific antibodies after transplantation is strongly associated with graft injury and loss and that incorporation of this risk factor improves risk stratification for graft loss.
Type of study : descriptive prospective cohort study
1.Summary of article- Presence of DSA is important and linked to inferior graft survival and antibody mediated rejection.But types of DSA ie whether it is complement binding or not, tell us better about graft survival and rejection.Presence of Complement binding DSA was associated with more acute rejection features like..Microvascular inflammation, Glomerulitis,Interstitial inflammation ,Presence of C4d in PTC as well as chronic rejection features like…Transplant glomeropathy,Interstitial fibrosis and tubular atrophy compared to DSA which are not complement binding. GFR at 1 yr was lowest in Complement binding DSA transplant compared to non complement binding DSA and No DSA transplant .Presence of Complement binding DSA drastically decrease graft survival compared to noncomplement binding DSA and No DSA transplant. Highlighting the fact that Presence of complement binding DSA may be more important than its quantity,,In this study Higher MFI was not associated with inferior graft loss compared to lesser values of MFI. 2. It was population based prospective study 3.Evidence level..2
Type of this study is a prospective cohort study Level of evidence is II The article proposed the issue of assessment of the complement-binding capacity of DSA which can be used to reflect the power of DSA and the impact on graft survival. Complement-fixing DSA can cause C4d positive ABMR, but it’s important to note that C4d deposition though specific but lacks sensitivity. Complement fixing DSA are more clinically significant than non-complement fixing ones, note that the level of MFI may not correlate well to the significance of the antibodies. The above study elucidates that ABMR occurs more commonly in patients with C1q-binding DSA (those had significantly lower graft survival than those with non-C1q-binding DSA’s) when compared to non-C1q-binding DSA although in which HLA antibodies had inferior graft survival than those without DSAs.
AMAL Anan
3 years ago
Summary of article :
The Road to HLA Antibody Evaluation: Do Not Rely on MFI :
– Technological advances in HLA laboratory testing undoubtedly improved the sensitivity and specificity of HLA antibody assessment but not without introducing a set of challenges
regarding data interpretation. In particular, the introduction of solid-phase single-antigen bead (SAB) antibody assessment brought the belief that mean fluorescence intensity (MFI) was a quantifiable value.
A transplant candidate with a calculated panel-reactive antibody value of 100% who lives in Georgia was recently offered a virtual cross-match– negative, HLA-nonidentical kidney from a deceased donor in California. With no detectable donor-specific antibodies (DSAs) in a current serum sample (>>Under Construction: The Journey Begins :
Advances in techniques and technology paved the way to improved testing. Enhancement of the CDC assay with antihuman globulin made it possible to detect lower levels of antibodies than had been previously feasible. Next, the advent of flow cytometry led to the discovery and clinical implementation of the flow cytometric cross-match. Once again, sensitivity to detect even lower levels of antibodies was enhanced with the added benefit of simultaneous and independent assessment of both T cell and B cell crossmatches.
>>>Hard Hats Required: Controversies in HLA Antibody Assessment :
While multiplex Luminex technology (Luminex Corporation, Austin, TX) has provided a specific and sensitive platform to identify HLA antibodies, it is not flawless. A major point of contention revolves around results from SAB testing being reported as a numerical value referred to as mean fluorescence intensity (MFI).
>>>New Directions :
With the introduction of more sensitive and specific approaches to determine donor– recipient compatibility, the HLA community has reaffirmed the power of pattern recognition. In the mid 1960s–1970s, the term “cross-reactive groups” (CREGs) was used to describe the serologic phenomenon of multiple unique HLA antigens that reacted with a single antibody. Rodey and Fuller reported that it was more common for patients to produce a few antibodies to shared determinants among a group of antigens than to generate multiple individual specificities .
>>> Conformation of an Integral Component to Antibody Pathogenicity :
While there has been a strong interest in the quantification of HLA antibodies, there has been virtually no attention given to how or whether antigen conformation affects antibody affinity, avidity, and resulting MFI values. The following example illustrates that it is reasonable to consider that the degree and strength of antibody binding is influenced by antigen conformation.
>>> Antigens Versus Antibodies :
It is important to realize that detecting DSAs is predicated on a two-sided equation. On one side is the patient’s serum (antibody); on the other, the antigen it recognizes. When a DSA is identified, it is crucial to understand that microparticles have been coated with an amount of antigen designed to detect low levels of antibody, not to mimic the quantity of antigen on lymphocytes or tissues.
>>> Merge: “Functional” Methods to Assess Antibody Pathogenicity :
Several studies have focused on the complement fixing characteristics of HLA antibodies,
anticipating that those properties would be associated with their pathogenicity. SAB assays
were modified to assess which, if any, HLA antibodies in patient sera bound specific complement components (C1q, C3d, C4d) as a surrogate of complement activation in vivo. Published studies have been inconsistent, with some reporting strong associations between complement fixation and poor allograft outcome , while others found no correlations.
>>> Guides to Antibody Assessment :
Difficult and problematic cases cannot always be resolved with additional testing and/or sophisticated algorithms. Sometimes, there are no other tests to perform. For example, identical antibody presentations (e.g. antibodies to HLA-A*02 well below the established cutoff value) in three different individuals being considered for transplantation with an HLA-A*02–positive donor do not mean the same pathway will be taken for all three subjects.
AMAL Anan
3 years ago
Please give a summary of this article :
– Anti-HLA antibodies hamper successful transplantation, and activation of the com- plement cascade is involved in antibody-mediated rejection. We investigated whether the complement-binding capacity of anti-HLA antibodies plays a role in kidney-allograft failure.
– Assessment of the complement-binding capacity of donor-specific anti-HLA antibod- ies appears to be useful in identifying patients at high risk for kidney-allograft loss.
– In the United States and Europe, thousands of kidney transplants fail each year, and kidney-allograft failure is a major cause of end-stage renal disease, leading to in- creased morbidity, mortality, and costs.
– Since the pioneering discovery in 1969 that anti-HLA antibodies are lymphocytotoxic,activation of the complement cascade has been con- sidered to be a key component of antibody-medi- ated allograft rejection, and C4d deposition in renal capillaries has been considered the foot- print of antibody-mediated allograft damage.
– The capacity of anti-HLA antibodies to bind complement fraction C1q, which is the first step in activation of the classic complement cascade, determines the cytotoxic potential of these anti- bodies, and an assessment of their complement- binding capacity may be useful both for risk strati- fication and for diagnosis of antibody-mediated rejection.
– The median follow-up after transplantation was 4.8 years (range, 0.2 to 7.0). The median follow- up times were 3.9 years (range, 0.4 to 7.0) in pa- tients with C1q-binding donor-specific anti-HLA antibodies and 4.3 years (range, 0.2 to 7.0) in pa- tients with non–C1q-binding donor-specific anti- HLA antibodies.
– The inclusion of complement-binding donor-spe- cific anti-HLA antibodies in the reference model significantly improved its discrimination capacity.
-In conclusion, we systematically evaluated im- munologic characteristics before and after trans- plantation in a population-based sample of kid- ney-allograft recipients, incorporating the full spectrum of graft phenotypes. We found that the presence of complement-binding anti-HLA donor- specific antibodies after transplantation is strongly associated with graft injury and loss and that in- corporation of this risk factor improves risk stratification for graft loss.
References
1. Nankivell BJ, Kuypers DR. Diagnosis and prevention of chronic kidney al- lograft loss. Lancet 2011;378:1428-37.
2. Gaston RS, Cecka JM, Kasiske BL, et al. Evidence for antibody-mediated injury as a major determinant of late kidney al- lograft failure. Transplantation 2010;90: 68-74.
3. Sellarés J, de Freitas DG, Mengel M, et al. Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonad- herence. Am J Transplant 2012;12:388-99.
4. Organ Procurement and Transplanta- tion Network and Scientific Registry of Transplant Recipients 2010 data report. Am J Transplant 2012;12:Suppl 1:1-156.
5. Rapport 2011 de l’activité de prélève- ment et de greffe de l’Agence de la Biomé- decine (http://www.agence-biomedecine. fr/Rapport-annuel-2011).
6. Terasaki PI, Ozawa M. Predicting kid- ney graft failure by HLA antibodies: a pro- spective trial. Am J Transplant 2004;4:438- 43.
7. Hourmant M, Cesbron-Gautier A, Terasaki PI, et al. Frequency and clinical implications of development of donor-spe- cific and non-donor-specific HLA anti- bodies after kidney transplantation. J Am Soc Nephrol 2005;16:280412.
AMAL Anan
3 years ago
What this type of this study?
-Prospective cohort study .
AMAL Anan
3 years ago
What is the level of evidence demonstrated by this study?
Level II evidence.
Manal Malik
3 years ago
1-Level of evidence:2
2-prospective cohort study.
3-Summary of complement-binding Anti -HLA Antibodies and kidney -Allograft Survival Introduction:
Complement–binding anti-HLA Antibodies mediated rejection is associated with a high risk for kidney allograft loss., so the alloimmune response is a major determinant of late kidney allograft loss.
The capacity of anti-HLA Abs to bind complement fraction c1q is the first step in activities of the classical complement cascade, leading to ABMR so the c4d deposits in renal capillaries. Methodology:
The study was carried out in Paris at Necker Hospital between January 1, 2005 andjan1,2011.
1016 patients in total.
ABOblood group compatible
CDCXM negative
Clinical data;
clinical data obtained from two national registries of Necker hospital are respected.
Documented cases of acute rejection according to the Banff criteria.
The protocol of treatment of allograft rejection was the same in all the 3 centers.
Histology&immuno-chemical tests:
Protocol-specified graft biopsies were performed 1 year after transplantation in 845 patients without any acute clinical rejection.
Specimens from biopsies were performed on 171 patients with acute allograft rejection.
Graft-biopsy were scored according to the updated Banff criteria
C4d stating was performed by immunochemical analysis on the paraffin section with the use of polyclonal antibodies
All patients were tested for the presence of circulating donor-specific anti-HLA antibodies in banked serum samples.
Statistical analysis:
The predictive value that C1q-binding status added to a reference risk model was evaluated with the use of the C-statistic.
Results for C1q binding DSA and allograft survival were replicated in the independent validation sample.
Results:
TCMR was seen in 14 of 77 patients with DSA & C1q binding capacity (18%).in 30 of 239 patients with DSA without c1q binding capacity (13%), and in 52 of 700 patients without DSA(7%).
C1q binding DSAs were associated with aggressive microvascular inflammation. Transplant glomerulopathy, high score C4d deposition in the PTC.this aggressive pathology was not seen in those with non-c1q binding DSA, and those without DSA
Patients with C1q-binding donor-specific anti-HLA antibodies had a lower (GFR) at 1 year than did patients with non–C1q-binding donor-specific anti-HLA antibodies and patients without donor-specific anti-HLA antibodies
Determinants of Kidney-Allograft Loss:
Complement-binding donor-specific anti-HLA antibodies remained independently associated with the risk of kidney-allograft loss after adjustment for the mean fluorescence intensity of donor-specific anti-HLA antibodies
Discussion:
C1q binding DSA was an independent predictor of graft loss more than 5 years Tx and clearly improved patient risk stratification for graft failure
Patients with C1q-binding DSA had more aggressive microvascular inflammation & transplant glomerulopathy
C1q binding DSA was associated with the risk of graft loss independent of MFI values
Regarding prognosis, C1q can identify pts at risk even with negative C4d
C4d, although specific, lacks sensitivity as an indicator of the humoral response.
There are therapeutic implications. Since promising therapeutic agents targeting complement (e.g., a C5 inhibitor [eculizumab] or a C1 inhibitor)
Limitation of the study:
Variability of MFI cut point between transplant centers, so it will not reflect the true result of this study Conclusion:
The presence of complement-binding anti-HLA donor-specific antibodies after transplantation is strongly associated with graft injury and loss.
Incorporation of this risk factor improves risk stratification for graft loss
It is a retrospective cohort study analyzing transplant patients from 2005 to 2011 in a French population of various transplant patients
Summary of the article: The study highlights the importance of the Complement binding anti HLA DSA in causing graft injury and ABMR. C4d in the renal biopsy of the transplant kidney has been considered the foot print of ABMR due to their persistence of C4d stain in the renal biopsy. Assays are available to detect C1q which is the first step in the complement pathway activation leading on to C4d degradation product which is available as a special stain in the renal biopsy. 1016 patients from 2 centers at France were included in the study. Immunosuppressive protocols and induction regimens were same across both the centers. They included all CDC T and B cell negative cross match patients and followed them up for 6 years. The SAB Luminex was tested on day 0 and after 1 year. 700 patients had no DSA. 239 patients had non complement binding DSA and 77 patients with complement binding DSA. Kidney biopsies were done 1 year later or whenever the clinical nature of rejection was suspected. They concluded that complement binding anti HLA DSA were associated with lowest graft survival at the end of 6 years (54%) as compared to 93% of those associated with non complement binding DSA. They had concluded that this cohort study could pave way for more clinical trails on the usage of complement inhibitors like C1q esterase inhibitors and ecluzimab for renal transplant as lowering the complement levels means less C1q binding and less severity of ABMR.
Asmaa Khudhur
3 years ago
One of the most important advances in trans- plantation medicine has been the recognition that anti-HLA antibodies are destructive.
Although anti-HLA antibodies are considered to be harmful, there is a wide spectrum of graft in- jury related to these antibodies, ranging from no recognizable damage to f lorid rejection.
activation of the complement cascade has been considered to be a key component of antibody-medi- ated allograft rejection, and C4d deposition in renal capillaries has been considered the foot- print of antibody-mediated allograft damage.
The capacity of anti-HLA antibodies to bind complement fraction C1q, which is the first step in activation of the classic complement cascade, determines the cytotoxic potential of these anti- bodies, and an assessment of their complement- binding capacity may be useful both for risk strati- fication and for diagnosis of antibody-mediated rejection.
We conducted a study to define the full spec-
trum of kidney-allograft injury according to the C1q-binding properties of donor-specific anti-HLA antibodies in a large population-based study and to determine whether assessment for the pres- ence of C1q-binding donor-specific anti-HLA anti- bodies after transplantation might improve risk stratification for kidney-allograft loss.
Results
Baseline Characteristics of the Kidney- Allograft Recipients
1016 patients undergoing renal transplantation were included in the main analysis. Three distinct populations were identified after transplantation, according to the presence or absence of donor-specific anti-HLA antibodies and complement-binding capacity: 700 patients without circulating donor-specific anti-HLA anti- bodies, 239 patients with non–complement-binding donor-specific anti-HLA antibodies, and 77 patients with complement-binding donor-specific anti-HLA antibodies.
Kidney-Allograft Injury
In the first year after transplantation, acute clin- ical rejection developed in 171 patients:
96 patients had T-cell–mediated rejection (56%) and 75 had antibody-mediated rejection (44%). T-cell– mediated rejection occurred in 14 of 77 patients with donor-specific anti-HLA antibodies plus C1q-binding capacity (18%),
in 30 of 239 patients with donor-specific anti-HLA antibodies without C1q-binding capacity (13%), and in 52 of 700 patients without donor-specific anti-HLA antibodies (7%) .Antibody-mediated rejection occurred in 37 patients with C1q-binding donor- specific anti-HLA antibodies (48%) and 38 patients with non–C1q-binding donor-specific anti-HLA antibodies (16%) .
Among the patients with C1q-binding donor- specific anti-HLA antibodies, 67 had microvas- cular inflammation (87%), 28 had tubular and interstitial inflammation scores of 2 or higher (on a scale of 0 to 3, with higher scores indicat- ing more severe abnormality) (36%), 18 had end- arteritis (23%), 17 had transplant glomerulopathy (22%), 30 had moderate-to-severe arteriosclerosis (39%), 23 had moderate-to-severe atrophy-scarring lesions (interstitial fibrosis and tubular atrophy) (30%), and 47 had C4d deposition in peritubular capillaries (61%). Patients with C1q-binding do- nor-specific anti-HLA antibodies had more ex- tensive microvascular inflammation and trans-
plant glomerulopathy and higher scores for graft peritubular capillary C4d deposition than both patients with non–C1q-binding donor-specific anti-HLA antibodies and patients without donor- specific anti-HLA antibodies .Stratified analyses revealed that these increases applied to samples from protocol-specified biopsies, per- formed at 1 year, and samples from biopsies performed during an acute-rejection episode in the first year .
Kidney-Allograft Survival
kidney-allograft survival according to donor-specific anti-HLA antibody status after transplantation. Patients with donor- specific anti-HLA antibodies had significantly worse graft survival than patients without donor- specific anti-HLA antibodies (5-year graft sur- vival after transplantation, 83% vs. 94%; P<0.001 by the log-rank test).
When patients with donor- specific anti-HLA antibodies after transplantation were subsequently categorized according to complement-binding capacity, patients with C1q- binding donor-specific anti-HLA antibodies had the poorest 5-year graft survival after transplantation (54%), as compared with patients with non– C1q-binding donor-specific anti-HLA antibodies and patients without donor-specific anti-HLA anti- bodies (93% and 94%, respectively. The risk of graft loss according to the donor-specific anti-HLA anti- bodies–C1q status at day 0 and the status after transplantation revealed that patients with C1q- binding donor-specific anti-HLA antibodies after transplantation had the highest risk of graft loss
Prediction of Kidney-Allograft Loss
the addition of complement-binding donor-specific anti-HLA antibodies to the reference model adequately re- classified patients at lower risk for graft loss and those at higher risk.
Discussion :
A sensitive detection method for C1q-binding anti-HLA antibodies yield findings in addition to those of the single-antigen flow bead test. Al- though the mean fluorescence intensity of donor- specific anti-HLA antibodies is widely used in clinical practice for risk stratification, this study
showed that C1q-binding donor-specific anti-HLA antibodies remained strongly associated with the risk of graft loss regardless of the mean fluorescence intensity of the antibodies.
C1q and C4d do not provide equivalent predictive information. C1q testing may help identify patients at risk, despite C4d .they found that C4d, although specific, lacks sensitivity as an indicator of humoral rejection. Assessment for complement-binding donor-specific anti-HLA antibodies may provide an early indication of the potential for complement-mediated injury, without the functional requirement for progression through the pathway to C4d deposition. Detection of complement-binding donor-specific anti-HLA antibodies may indicate which of the antibodies present have the capacity to activate the complement cascade,
potentially but not inevitably leading to C4d de- position and complement-mediated damage or antibody-mediated injury in vivo. Such findings do not rule out a possible role for complement- independent mechanisms in allograft injury me- diated by complement-binding donor-specific anti- HLA antibodies.
Type of study : descriptive prospective cohort study with analytic component
Level of evidence: II
Ben Lomatayo
3 years ago
The Road to HLA anti-body Evaluation: Do Not Rely on MFI
Introduction In past CDCXM was the available tests before transplantation. It has less specificity( pts with +Ve test may have good outcome) and less sensitivity( pts with negative test may still have graft failure) .Therefore, more reliable test was needed to detect & identify clinically important HLA Abs
Under Construction : The Journey Begins
CDCXM enhancement ; e.g. addition of anti-globulin
FCXM; more sensitive than CDCXM
Micro-particle technology; cell-sized latex or polystyrene beads; A game changer in detection & identification of HLA Abs. They include Flow PRA TM(screening) and SAB assays e.g One lambda( semi-quantitative)
Hard Hats Required : Controversies in HLA Antibody Assessment
SAB read out is expressed as MFI which gives numerical value
MFI was not intended to be quantitative and Luminex platform was not approved by FDA as a quantitative assay[2]
Despite this facts,MFI is still being used as a semi-qunatitative assessments of antibody by both laboratorians and clinicians
If MFI is decreasing after after treatment of AMR it is considered as a good clinical response and if no change or raising it is viewed as treatment failure
so what are the implications of depending completely on MFI values to make therapeutic decisions ?
Be Prepared to Stop : Limitations
The problem is what is the MFI threshold for positive beads or negative beads ?
If the MFI threshold is too low = false positive results . This will prevent patients from receiving compatible organ
If the threshold too high = false negatives results. This will lead to early AMR
Some patients transplanted across very low MFI = 100 and they developed poor graft survival, while other patients whom were transplanted across very high MFI =10,000 enjoyed excellent graft survival[3,4]
Therefore, MFI cut off value values can vary by the transplanted organ and/or transplant center practice
Causes of MFI variability ; 1. Vendor platform 2.Antigen source 3. Reagents 4. Intra-laboratory [5](This can be > 50% due to the technician = pipetting , washing, vortex speed, centrifuging and day to day variability = temperature, PH changes of the reagents)
other factors that affects MFI values are ; 1. Inferences from complements e.g.C1q [7] 2. Autoantibodies e.g. IgM [9] both can lead to false negative results 3. Factors related to manufacturing & coating of the micro-particles
False positive results can occur due to ; 1. antibodies against misfolded proteins( denatured antigens) 2. Antibodies against cryptic antigens [10] ( antigens there are normally antibody in-accessible) . These antibodies are more prevalent among wait list patients 11 -40% and this explained why some patients with high MFI-DSA do not experience poor graft outcomes.
Few studies showed that antibodies against dentures antigens does not associated with poor outcomes[11]
Antibodies to both native & denature antigens of the same specificity may be behave differently [13]. There is early to consider antibodies against denture antigens as clinically innocuous.
Re-routing : New Directions
‘Cross-reactive groups’ (CREGs)
Old terminology
Introduced in 1960s- 1970s
Serologic phenomenon ; Single antibody bind to a group of antigens
Example ; HLA-A23, -A24, -A68, -A69, HLA-B75, HLA-B58 all are HLA-A2 CREGs. Therefore antibody to HLA-A2 will cross react to all of the above mentioned HLA-A2 CREGs
The new terminology are ; Epitopes, Eplets, Conformational Variance which represent the current version of CREGs
Examples ; The 62GE epitope is expressed by HLA-A2, HLA-B57,& HLA-B58 and the 127K epitope is expressed by HLA-A2,-A23,-A24, -A68, and -A69
The current trends is to change bead reactivity from antigen to epitope
Bumps in the road : Conformation – An integral component to antibody pathogenicity ?
Antigen conformation may affect antibody affinity,avidity, and MFI values.
The degree & strength of antibody binding is affected by antigen conformation
Change in amino acid sequences or positions are the bases of antigen conformation and this will affect the MFI value
Divided Highway : Antigens versus Antibodies
DSA that binds a beads in Vitro may fail to bind the corresponding antiden inVivo because it is expression is low. However,antigen expression in viv may increase in inflammatory conditions and the allograft may be susciptible to antibody binding.
Antibody can diluted over many beads sharing the same epitopes(public epitopes). This can weaken the antibody strength and lead to negative MFI or weak fluorescence signal
Therefore, the pathogenicity of an antibody cannot and should not be assumed based completely on MFI values
Merge: ‘Functional’ methods to assess antibody pathogenicity
These are complement proteins ; C1q, C3d, C4d
It is controversial as some studies revealed associations between complement fixation and poor allograft outcome[17-19], while others did not find any correlation[20,21]
One study of the complement-fixting DSAs reported clinical relevant only in the post-transplant period[17]
The IgG1 DSAs the most abundant subclass often correlates with normal allograft function[23,24] indicating that IgGs that fix complement in Vitro may not do so in Vivo due to ; 1). the presence of complement inhibitor in the serum e.g. heparin suphate[26] 2). the presence of high levels of decay-accelerating factor(DAF)[27] 3). Hexamer formation( hexamerisation of IgG Fc fragments is required to initiate and activate complement cascade)
AMR may occur with mechanism other than complement involvements e.g. smooth muscle cell, endothelial cell activation & antibody-dependent cellular cytotoxicity via macrophages & NK cells[29]
Re-paving the Road : Guide to Antibody Assessment
Team approach is required in making decisions regarding challenging cases[30]
This involve face -to -face conversation between transplant Nephrologist, transplant surgeons, immunologists , transplant coordinators
The objective is to outline the immunological complexities & to discuss issues typically one or more of the team members do not consider
Through this inter-actions institutional practices are fine-tuned to best fit patient need.
Conclusion : There is long way to go regarding HLA antibody detection and identification New tests are needed but it is still not clear if these test will answer the old and future questions in this crucial matter in transplantation
CARLOS TADEU LEONIDIO
3 years ago
1 – RESUME:
Complement-Binding Anti-HLA Antibodies and Kidney-Allograft Survival
Various studies over the past decade have indicated that the alloimmune response, mediated by anti-HLA antibodies, plays a key role in the failure of kidney allografts; this concept has been extended to heart, lung, and composite tissue transplants. Such a varied effect underscores the need to define distinct graft phenotypes and outcomes according to the presence or absence and characteristics of donor-specific anti-HLA antibodies after transplantation. Anti-HLA antibodies are lymphocytotoxic, activation of the complement cascade has been considered to be a key component of antibody-mediated allograft rejection, and C4d deposition in renal capillaries has been considered the footprint of antibody-mediated allograft damage. The capacity of anti-HLA antibodies to bind complement fraction C1q, which is the first step in activation of the classic complement cascade, determines the cytotoxic potential of these antibodies, and an assessment of their complementbinding capacity may be useful both for risk stratification and for diagnosis of antibody-mediated rejection.
This article is about one study in Paris at Necker Hospital and Saint-Louis Hospital between January 1, 2005, and January 1, 2011. The transplantation allocation system was identical for the three centers and followed the rules of the French national agency for organ procurement (Agence de la Biomédecine). All transplants were compatible with the ABO blood group. A negative result of cross-matching for IgG T-cell and B-cell complement-dependent cytotoxicity was required for all recipientes. It documented all cases of acute clinical rejection, defined by deterioration in graft function, proteinuria, or impaired function and histopathological evidence of rejection, according to the consensus rules of the international Banff classification criteria.
RESULTS:
Three distinct populations were identified after transplantation:
– patients without circulating donor-specific anti-HLA antibodies – 700
– patients with non–complement-binding donor-specific anti-HLA antibodies – 239
– patients with complement-binding donor-specific anti-HLA antibodies – 77
1 – Kidney-Allograft Injury
In the first year after transplantation, acute clinical rejection developed in 171 patients:
– 96 patients had T-cell–mediated rejection (56%)
– 75 had antibody-mediated rejection ( 44%)
– Patients with C1q-binding donor-specific anti-HLA antibodies had more extensive microvascular inflammation and transplant glomerulopathy and higher scores for graft peritubular capillary C4d deposition than both patients with non–C1q-binding donor-specific anti-HLA antibodies and patients without donor specific anti-HLA antibodies
– Patients with C1q-binding donor-specific antiHLA antibodies had a lower estimated glomerular filtration rate than did patients with non–C1q-binding donor-specific anti-HLA antibodies and patients without donor-specific anti-HLA antibodies
2 – Kidney-Allograft Survival
The median followup times were 3.9 years in patients with C1q-binding donor-specific anti-HLA antibodies and 4.3 years in patients with non–C1q-binding donor-specific antiHLA antibodies.
Patients with donor specific anti-HLA antibodies had significantly worse graft survival than patients without donor specific anti-HLA antibodies.
3 – Determinants of Kidney-Allograft Loss
The following independent predictors of graft loss were identified:
– low estimated GFR at 1 year:
– interstitial fibrosis and tubular atrophy
– glomerular and peritubular inflammation and transplant glomerulopathy
– presence of complement-binding donor specific anti-HLA antibodies after transplantation
DISCUSSION
Was observed that the presence of complement-binding donor-specific antiHLA antibodies detected in the first year after transplantation was an independent predictor of kidney-allograft loss more than 5 years after transplantation and significantly improved individual risk stratification for graft failure
2 – What is the level of evidence demonstrated by this study?
Evidence level 2
3 – What this type of this study? This study is a prospective cohort
Zahid Nabi
3 years ago
Development of Anti HLA antibodies leads to a poor graft outcome and whether complement binding adds to this deleterious affect has been investigated in this perspective cohort study of Alexander loupy and colleagues.
This was a multi center study conducted in Paris. A total of 1016 Patients were enrolled between January 1,2005 and January 1, 2011.
The participants were divided into two groups. As an external validation cohort, beneficiaries of organ transplants from a different transplant hospital were recruited. The anti-HLA antibodies and complement binding capabilities of all the patients were tested on days 0 and 1-year post-transplant, as well as during the moment of acute clinical rejection, in order to identify donor-specific antibodies. A biopsy of the kidney graft was performed one year after the transplant, or at the time of acute clinical rejection.
In the first year 171 patients developed acute clinical rejection, 96 patients TCMR and 75 ABMR.
TCMR patients were as follow: 14/77 with DSAs with C1q binding capacity, 30/239 with DSAs without C1q binding capacity, 52/700 without DSAs.
ABMR patients were as follow: 37/77patients with DSAs with C1q binding capacity, 38/239 patients with DSAs without C1q binding capacity.
Patients with DSAs with C1q binding capacity had increased risk of extensive microvascular inflammation and transplant glomerulopathy and higher scores for graft peritubular capillary C4d deposition and lower estimated GFR than both patients with non–C1q-binding donor-specific anti HLA antibodies and patients without donor specific anti-HLA antibodies.
C1q binding DSA (48%) was associated with severe ABMR than non-complement fixing antibodies(16%) and 5 yr Graft survival was less in patients with C1q binding DSA ( 54%) than non C1q binding DSA ( 93%) . Other factors that worsened graft outcome included increment in donor age, each one minute increase in cold ischemia time, deceased donor and retransplant Therapeutic agents targeting complement (e.g., a C5 inhibitor [eculizumab] or a C1 inhibitor) are being used in transplant recipients
Conclusion: the presence of DSA is associated with poor allograft outcome and also helps in risk stratification of patients for graft loss .
Despite huge advances in transplantation, alloimmune response still the main factor associated with late graft loss[1-3]
Alloimmune response is mediated by anti-HLA antibodies which causes rejection and graft failure[11-12]
This study investigated if the complement binding capacity of anti-HLA antibodies can result in allograft failure
Methodology ;
Study population ; After approval & obtained written concern
This study was carried in Paris at Necker Hospital, Saint-Louis Hospital between Jan 1,2005 and Jan 1,2011, 1016 patents in total
External-validation cohort was considered at Foch hospital in Suresnes between Jan 1,2004 and Jan 31, 2010
ABC compatible
CDCXM negative
Clinical data ;
This were taken from national registries of Necker hospital, Sain Louis and Foch hospitals
Includes ; documented cases of acute rejection , histopathological evidence of rejection based on Banff criteria[27,28]
Treatment of allograft rejection was the same in all of the 3 centers
Histologic & immuno-chemical tests ;
Protocol biopsies at 1 year for 845 patients and biopsies for acute rejection in 171 patients
Biopsies were classified according to Banff criteria [27,28] by 3 well trainied pathologist
C4d staining was done by immunohisto-chemistery on parafin section with addition of anti-C4d antibodies
Detection & Characterization of Donor-specific anti-bodies ;
DSA was tested in banked serum sample by Luminex platform (one Lambda)and in serum sample collected at the time of biopsy at 1 year after transplantation
DSA were evaluated in a blinded way at University of Pittsburgh for the presence of C1q- binding capacity with One Lambda [17,18,21]
Statistical analysis ;
The association of clinical,histologic ,functional, and immunologic factors with graft loss was evaluated in uni-variate & multivariate cox regression analysis
The predictive value that C1-binding status added to a reference risk model was evaluated with the use of C-statistic[32-33]
Results for C1q binding DSA and allograft survival were replicated in the independent validation sample
Results ;
TCMR was seen in 14 of 77 patients with DSA & C1q binding capacity(18%), in 30 of 239 patients with DSA without C1q binding capacity(13%), and in 52 of 700 patients without DSA(7%)
AMR developed in 37 pts with C1q binding DSA(48%), and 38 pts with non-C1q binding DSA(16%)
C1q binding DSAs were associated aggressive microvascular inflammation, transplant glomerulopathy, high score C4d deposition in the ptc. This aggressive pathology was not seen in those with non-C1q binding DSA, and those without DSA.
Lower eGFR 42 -/+ 22 ml/min was observed in in pts with C1q binding DSA than those with non-C1 binding DSA and those without DSA
Groups with C1q binding DSA experienced the worst 5 years graft survival (54%) than those with non-C1q binding DSA(93%) and those without DSA(94%)
Determinant of kidney allograft loss ;
The independent predictors of graft loss were ;
Low eGFR at 1 year ( hazard ratio 12.49)
Interstitial fibrosis & tubular atrophy(hazard ratio 2.22)
Glomerlular & peritubular infammmation(hazard ratio 2.26)
Transplant glomerulopathy(hazard ratio 2.26)
C1q binding DSA after Tx(hazard ratio 4.78)
Discussion ;
C1q binding DSA was independent predictor graft loss more than 5 years Tx and clearly improve patient risk stratification for graft failure
Patients with C1q-binding DSA had more aggressive microvascular inflammation & transplant glomerulopathy
C1q binding DSA was associated with risk of graft loss independent of MFI values
Regarding prognosis C1q can identify pts at risk even with negative C4d
C4d athough specicfic , lacks sensitivity as an indicator of humoral response[36,37]
Limitations of the study ;
The study was not design to evaluate effect of treatment on the C1qbinding-DSA or to provide kinetics of the capacity of C1q binding of DSA.
Conclusion ;
This study found that C1q binding DSA after Tx is strongly associated with graft injury & loss
The incorporation of this risk factor improves risk stratification for graft loss.
Level 4
Population based study
Reem Younis
3 years ago
. The Road to HLA Antibody Evaluation: Do Not Rely on MFI
FlowPRA™ and SAB assays are used for the detection of HLA antibodies. FlowPRA™ is a screening test, one that qualitatively detects the presence/absence of HLA antibodies. SAB is a semiquantitative test used to determine antibody specificity.
-The results from SAB testing report as a numerical value referred to as mean fluorescence intensity (MFI). MFI values were never intended to quantify antibodies, nor was the Luminex-based test approved as a quantitative assay by the US Food and Drug Administration.
-MFI values reflect a given bead’s relative fluorescence without reference to a standard and fluorescence can be affected by many variables.
– The tendency is to correlate MFI values with clinical outcomes and to serially monitor their fluctuations as a measure of clinical status. Decreasing MFI values after a patient has been treated for antibody-mediated rejection (AMR) is interpreted as a proper clinical response, while no change (or an increase in value) is considered a therapy failure.
-If the MFI threshold is set too low, false positives may preclude a patient from receiving a compatible organ, while if the threshold is set too high, there may be an increased risk that a patient will experience early and/or irreversible AMR.
-There is no single value of MFI that corresponds to an unequivocal threshold above or below which organ transplantation is contraindicated or risk-free, respectively. As a result, cutoff MFI values can vary significantly by the organ transplanted and/or a transplant program’s practice.
-MFI variability is compounded by vendor, antigen source, test reagents, antibody-dependent complement activation, and intralaboratory variability.
– Mechanisms of assay interference include IgM HLA antibodies, wherein such antibodies compete with or block the binding of IgG HLA antibodies.
– False-positive reactivity can occur due to antibody binding to misfolded HLA proteins, commonly referred to as denatured antigens.
– Reactivity is attributed to the binding of antibodies to so-called cryptic epitopes, which under normal circumstances are considered to be antibody inaccessible. Such antibodies have a high prevalence among waitlist candidates (11–40%) likely explaining why some high-MFI DSAs were not associated with poor graft outcomes.
-Cross-reactive groups” (CREGs) were used to describe the serologic phenomenon of multiple unique HLA antigens that reacted with a single antibody.
– The degree and strength of antibody binding is influenced by antigen conformation.
– Microparticles have been coated with an amount of antigen designed to detect low levels of antibody.
-The most common de novo antibody produced posttransplantation is directed to HLA-DQ antigens (15) despite low expression of the DQ loci.
-The “pathogenicity” of an antibody cannot and should not be assumed based strictly on MFI levels.
– There are associations between IgG DSA subclass (in particular, IgG3) and poor clinical outcomes in the posttransplantation setting.
-A better understanding of the processes that drive alloantibody production in an individual is necessary to best characterize the pathogenicity of a given antibody.
Alaa eddin salamah
3 years ago
The Road to HLA Antibody Evaluation: Do Not Rely on MFI
This article describes the techniques and technologies of HLA Abs testing starting from CDC enhancement with antihuman globulins to detect lower levels of Abs and the development of flow cytometry crossmatch techniques till the breakthrough of microparticles technologies introduction to practice.
Controversies in HLA Antibody Assessment
Although, multiple Luminex technology provided high specific and sensitive test for HLA Abs its report in numerical value as MFI become a mislead to clinicians’ interpretation of the results. The presentation of MFI as a numerical value gives the impression that this is a quantitative test – which is not-. Actually, MFI reflects the relative fluorescence of a given bead without a reference to a standard.
Limitations
MFI thresholds are variable between transplant centers and depending on a single test like MFI.if a given center chose a low threshold In order to decrease graft rejection the patient is prevented from receiving a compatible donor, in actual practice transplanted kidneys with very low threshold of MFI experienced poor graft outcome. On the other hand, transplanted patients with very high MFI threshold had excellent graft outcome!
So, why to sit these rigid cutoffs if their reflections on clinical outcome is not practical!
MFI variabilities can be caused by
1- Intra-laboratory variabilities caused by day-day variabilities including ambient temperature
2- Technologists dependent actions like pipetting, washing…etc.
3- Interference due to antibody-dependent complement activation in which the compliment split products bind to Ag-Ab complexes on the beads blocking secondary Abs binding resulting in diminished MFI values>> this may result on graft loss reported with low MFI
4- IgM HLA Abs competing against IgG HLA Abs binding sites cause the same results in point 3
5- Literature reported that sera from some patients contain Abs against both native and denatured HLA Ags (these denatured Ags result from manufacturing and coating of microparticles), normally Abs do not interact with these misfolded HLA but they are detected on the test resulting in high MFI>> this may explain why some patients with high MFI DSAs were not associated with poor graft outcome.
New Directions
CREG term introduced in 1960s to describe the phenomena of a single Ab reacts to multiple unique HLA Ags. Today, this observation was illustrated by Abs reaction to epitopes, eplets and conformational variances. We can consider Abs reacting to epitopes rather than HLA Ags this can lead to interpret non- DSAs as DSAs. (The target of DSAs is epitopes rather a whole HLA Ag)
Conformation—An Integral Component to Antibody Pathogenicity?
Actually, no attention is given on how Ag conformation affects Abs affinity, avidity and the resulting MFI values.
For example, Ab to Bw6 with high MFI except for B*46:01- and B*73:01-bearing beads which they have a single amino acid substitution in their epitops at position 75 (from glutamic acid to valine), interestingly HLA-C locus Ag had the same substitution (sharing Bw6/V75 epitop) resulting in low MFI also. This is an example of a single amino acid but this may be cause by several amino acids differences. Whatever the change, it collectively results in a physical alteration that affect MFI values.
Antigens Versus Antibodies
DSAs are detected by the interaction of the patient’s Abs with the Ags expressed in microparticles (which are designed to detect low titr Abs) but they are not expressive of the actual Ags load on lymphocytes or tissues. So, reacting Abs in vitro are not importantly representative of their interaction in vivo -although, inciting events like inflammation may renders the transplanted organ more susceptible to Abs binding. In conclusion, the pathogenicity of Abs cannot and should not be assessed solely on MFI values.
Functional” Methods to Assess Antibody Pathogenicity
Back to previous section, the pathogenicity of Abs may be assessed more practically by the Abs compliment fixing characteristics. So, SAB assays were modified to assess if Abs can bound compliment components (C1q, C3d, C4d) as a surrogate of complement activation in vivo. The results are debatable, were some proposed strong correlation and others no correlation at all. One large study, proposed that complement fixing DSAs were of clinical significance only on the posttransplant period.
Guides to Antibody Assessment
Each case should be assessed separately, Difficult cases cannot be assessed straightforward with algorithms or a single correct approach. The issue of HLA Abs detection, identification and interaction is still evoluting and may be or may be not one day evolute to the point where to reflect the patient’s true clinical interaction and outcome
Hinda Hassan
3 years ago
Please give a summary of this article
Not all anti-HLA antibodies are associated with graft injury. This study hypothesized that their ability to bind complement plays a major role in their significance in causing graft injury. This study included 1016 patients and was conducted in two transplantation centers in Paris from 2005till 2011 . Screening of circulating DSA anti-HLA antibodies , their complement-binding capacity and graft injury phenotype were assessed. Complement binding DSA were significantly associated with less 5-year graft survival, high AMR , severe form of graft injury and more c4d deposition, compared to non–complement-binding DSA anti-HLA antibodies and patients without DSA. C1q-binding donor-specific anti-HLAantibodies remained strongly associated with the risk of graft loss regardless of the meanfluorescence intensity of the antibodies.This study postulated that the detection of complement-binding DSA anti- HLA antibodies in the first year post transplantation is an independent predictor of kidney-allograft loss more than 5 years after transplantation and significantly improved individual risk stratification for graft failure.
What is the level of evidence demonstrated by this study?
2b
What this type of this study?
cohort
Mahmud Islam
3 years ago
The ROAD to HLAantibody evaluation: Do not rely on MFI
In this article, the advancement of technology and antibody detection was discussed. especially with the new solid-phase assay technology single antigen detection had very important information valuable in risk assessment. with this advancement in contrary to classic PRA evaluations even overseas or distant matching became available. although the mean fluorescence intensity (MFI) used here was not intended to be a quantitative measurement in practice it was sealed as such. there is variability from center to center and the other is a handicap. some antibodies with low intensity may disappear as they bind to multiple beads to become undetectable or detected but are considered as non-significant as they are below the threshold.
The problem with MFI is that if the threshold is set to a very low level this may preclude compatible patients from receiving organs. on the contrary setting, it is too high will lead to increased rejection.
the phenomenon of cross-reactive groups described in the 1960s-1970s now describes as eplets, epitopes is another challenge. public epitopes shared between multiple HLA antigens will lead to rejection although not presented in the report or may be presented with low intensity…
another poşnt is the relation between AB-AG; in the test, the beads are coated to detect very low levels of antibodies which may not be the parallel case in vivo and vice versa.
The type of IgG is also important. In vitro IgG1 and IgG3 fix complement to a higher degree (Ig2 less, IgG4 may not). In vive the same extent of effect may not bee seen. Hexamerisation of IgG Fc fragment and engagement of IgG antibodies may not correlate with their concentration. presence of IgG antibodies may not be sufficient to sustain or to start the complement activation cascade.
same antibody even with similar concentrations but in different recepients is not expected to have same effect.
Weam Elnazer
3 years ago
An overview of this article.
Graft damage is related to the presence of anti-HLA antibodies. It has been shown that they bind to the C1q component of complement. In order to determine the function of complement binding capability of anti-HLA antibodies in renal graft prognosis, an open-label, prospective cohort research was designed and implemented.
A total of 1016 transplant patients from two transplant facilities in Paris were involved in the research between January 2005 and January 2011. The participants were divided into two groups. As an external validation cohort, beneficiaries of organ transplants from a different transplant hospital were recruited. The anti-HLA antibodies and complement binding capabilities of all the patients were tested on days 0 and 1-year post-transplant, as well as during the moment of acute clinical rejection, in order to identify donor-specific antibodies. A biopsy of the kidney graft was performed one year after the transplant, or at the time of acute clinical rejection.
The existence of DSAs that bind to C1q in the sera of patients with DSA was investigated.
Patients were selected based on the presence or absence of DSA as well as their complement binding capability.
Detection of complement binding DSA in the first year after transplantation was an independent predictor of graft loss 5 years after transplantation and considerably improved risk stratification for graft failure, according to the study.
Those that suffered from complement binding DSA had graft damage, which was characterized by microvascular inflammation and C4d deposition.
The presence of C1q binding DSA was shown to be significantly linked with the likelihood of graft loss, independent of the MFI of the antibodies.
The detection of complement binding DSA may suggest the possibility of complement-mediated damage before development along the route leading to C4d deposition in the body.
The capacity to bind complement may be essential since novel therapeutic drugs such as the C5 inhibitor (eculizumab) and the C1 inhibitor (adalimumab) that target complement are increasingly being utilized in kidney transplant patients.
The type of the study: a prospective cohort study with a defined sample size the level of evidence: Evidence at the second level
Riham Marzouk
3 years ago
summary of additional article
MFI mean fluorescence intensity has some limitations make it not a good indicator for transplantability;
It has no universal cut off value , above which there is a contraindication for transplantation, as it is dynamic vary from day to day and from center to center, it is of value in showing response to treatment of rejection.
There are a cases transplanted against high MFI showed favorable outcome with no episode of rejection, and other cases are transplanted with low MFI showed occurrence of episodes of rejection with non-favorable graft outcome.
Interlaboratory factors can lead to variable MFI accounts for more than 50% of its variability, it is affected by reagent used, kits itself, daily variation of MFI and also technologist worked (operator dependent), all interlaboratory factors can lead to false positive of false high MFI which negatively impacts the decision of transplantation.
We should be epitope minded
CREGs cross reactive groups described reactivity of multiple HLA antigens with single antibody.
Rejected transplanted graft happens even with matched HLA antigen , this will be clarified by shared antigen in single antibody , so the match should be directed to the level above the level of antigen like epitope (specific epitope ) which will improve graft survival and outcome.
Amit Sharma
3 years ago
The road to HLA antibody evaluation: Do not rely on MFI
Introduction:
HLA antibody testing has undergone a sea-change in last 5 decades. From requirement of a negative crossmatch for transplant, we have reached a stage where transplants are being performed without a physical crossmatch. The journey of development of antibody testing is an interesting one.
Under construction: The journey begins.
The sensitivity of the original CDC crossmatch increased on using antihuman globulin. The sensitivity of antibody testing increased by use of flowcytometry and introduction of microparticle technology (FlowPRA and single antigen bead/ Luminex) further enhanced both sensitivity and specificity.
Hard hats required: controversies in HLA antibody assessment
MFI values provided by the Luminex technology give a semiquantitative assessment of antibody levels, signifying a relative fluorescence of a single bead after binding to antibody. MFI values do not correlate with clinical outcomes.
Be prepared to stop: limitations
The limitations of MFI include lack of clarity with respect to cut-off values; lack of standardization with respect to vendor, source of antigen and test reagents as well as intralaboratory differences. Various factors interfering with the assay include IgM HLA antibodies and complement split products giving rise to falsely low MFI, as well as denatured antigen giving rise to false positive results.
Rerouting: New directions
The power of pattern-recognition is important in antibody detection, represented by CREGs (cross-reactive groups) – multiple HLA antigens reacting with a single antibody, nowadays called epitopes, eplets etc. So, bead reactivity should be interpreted with respect to epitopes and not antigens.
Bumps in the road: Conformation – an integral component to antibody pathogenicity?
The avidity and degree of antibody binding to antigen depends on the conformation/ physical alteration of the antigen leading to variable MFI levels with same antibody, making it very difficult to interpret the results. hence eplets (discontinuous polymorphic regions) are important part of compatibility testing.
Divided highways: antigens versus antibodies
DSA detected by antigen binding in vitro might not bind to the antigen in vivo due to altered expression levels of the antigen, but might bind once the antigen expression increases as after an inflammatory stimulus. So, the MFI levels cannot be a marker of pathogenicity of the antibody.
Merge: “functional” methods to assess antibody pathogenicity
SAB assays identifying complement binding antibodies did not conclusively prove their association with poor outcomes, especially in pre-transplant period. Post-transplant IgG3 antibodies have been shown to be associated with poor graft outcomes. IgG1 DSAs have been shown to have good graft outcomes. IgG may not fix complement in vivo due to presence of complement inhibitors like heparin sulphate and DAF, non-hexamerization of Fc fragment and insufficient IgG molecule binding.
Repaving the road: guide to antibody assessment
Each case is different and center-specific multidisciplinary approaches for kidney transplantation is need of the day. HLA antibody detection and interpretation of its results needs to be done in light of the history and clinical parameters of the patient.
mai shawky
3 years ago
· summary of additional article about MFI
Although Luminex SAB is the standard used in HLA typing, many limitations exist.
· SAB and MFI were never designed to quantify DSA. However, their trend can be followed to determine response to desensitization protocol or anti-rejection therapy in AMR.
· Q. why not to use MFI as indicator for translantability (CI to transplant or predictive for the prognosis)?
o No defined cut off level for +ve or -ve tests (not standardized between different centers).
o Low threshold may hinder transplantability.
o Higher threshold may carry risk of early AMR.
o No direct relation was found between MFI and graft outcome. However, cases with low MFI had poor prognosis and others with higher MFI survived well.
o Marked intra-laboratory differences depending up on:
§ Temp and PH of reaction.
§ Methodology and amount of fluroscenated antibodies added.
§ In case of complement fixation antibody reaction (complement binding may decrease or interfere with anti HLA IgG antibodies (false -ve results).
§ Presence of IgM antibodies and anti-epitopes antibodies can give positive results invitro. However, they don’t usually reflected on in-vivo reaction and graft outcome.
§ Recent trend toward anti-epitope antibodies.
§ Reaction to bead coating with antigen differ from in-vivo reaction with lymphocytes antigens, so +ve DSA in-vitro may fail to react with cells in-vivo.
§ Although complement fixing antibodies ( IgG1, 3 ) were thought to have worse prognosis and early occurrence in early post transplant period than non complement fixing (IgG 2, 4 antibodies), some reports show conflicting results. This can be explained by the difference in reaction between invitro testing and actual invivo immune reaction.
· So , no single best test and no single approach in antibody testing..
· Individualized approach and case by case management is the ideal approach.
· Never to depend on snapshot to determine recipient risk, instead, tailor treatment to history, previous transplant and prior sensitization.
Alaa eddin salamah
3 years ago
Complement-Binding Anti-HLA Antibodies and Kidney-Allograft Survival
Introduction
Alloimmune response is a major cause of renal transplant failure. Alloimmune response mediated by anti-HLA Abs play a key role in failure of kidney graft and these Abs response varies between no effect at all to destructive outcomes on the allograft.
Anti HLA Abs are lymphocytotoxic and cause the activation of compliment cascade by binding to C1q – which is the first step in activating the classic pathway- leading to ABMR, this is why C4d deposits in renal capillaries is a landmark of ABMR.
This study purpose was to asses if C1q binding properties of DSAa may improve risk stratification for kidney-allograft loss.
Methodology
The data collected prospectively from three different centers with all patients are ABO compatible and with negative cross match for IgG T cell and B cell complement dependent cytotoxicity and patients were followed at 66 months, 1 year and every year after for a total of five years.
Acute clinical rejection was defined by deterioration in graft function, proteinuria, or impaired function and histopathological evidence of rejection, according to the consensus rules of the international Banff classification criteria. Immunosuppression protocols and treatment of allograft rejection episodes after transplantation were similar among the centers.
854 biopsies were taken from patients with no evidence of acute rejection at 1 year and 171 biopsies were taken within 1 year from patients with evidence of acute rejection. All biopsies were categorized by Banff score by three different pathologists who were blinded to the status of patients’ HLA As, C1q bonding status and clinical score. Also, C3d staining was performed by immunochemical analysis.
Detection and Characterization of Donor-Specific Antibodies
patients were tested for the presence of DSAs at day 0 and at time of biopsy (either at 1 year or at time of rejection in the first year) using single Ag flow bead assays on Luminex platform. Patients with identifiable DSAs were analyzed for the presence of C1q-binding donor-specific anti-HLA antibodies with the use of single-antigen flow bead assays
Results
Baseline Characteristics of the Kidney-Allograft Recipients
A total of 1016 patients were included at the time of transplantation in three groups
1- Without circulating DSAs n=700
2- Non complement binding DSAs 239
3- Complement binding DSAs 77
Kidney-Allograft Injury
In the first year 171 patients developed acute clinical rejection, 96 patients TCMR and 75 ABMR.
TCMR patients were as follow: 14/77 with DSAs with C1q binding capacity, 30/239 with DSAs without C1q binding capacity, 52/700 without DSAs.
ABMR patients were as follow: 37/77patients with DSAs with C1q binding capacity, 38/239 patients with DSAs without C1q binding capacity.
Patients with DSAs with C1q binding capacity had increased risk of extensive microvascular inflammation and transplant glomerulopathy and higher scores for graft peritubular capillary C4d deposition and lower estimated GFR than both patients with non–C1q-binding donor-specific anti HLA antibodies and patients without donor specific anti-HLA antibodies.
Kidney-Allograft Survival
Patients with DSAs with C1q binding capacity had the poorest rates of 5-year graft survival followed by DSAs without C1q binding capacity and patients without DSAs.
Determinants of Kidney-Allograft Loss
Independent predictors of graft loss yielded from analysis include: low estimated GFR at 1-year, interstitial fibrosis and tubular atrophy, glomerular and peritubular inflammation and transplant glomerulopathy, and the presence of complement-binding donor specific anti-HLA antibodies after transplantation (theses DSAs were associated with the same graft survival regardless of their MFI)
Sensitivity Analysis
At both centers, patients with complement binding DSAs had the lowest rate of graft survival, complement-binding donor-specific anti-HLA antibodies after transplantation remained independently associated with graft loss whether they were detected in specimens from protocol-specified biopsies performed at 1 year or in specimens from biopsies performed during acute rejection in the first year, C1q-binding DSAs C1q-binding
Discussion
The presence of complement binding DSAs in the first year after transplant were independently associated with allograft loss at 5 years follow up and significantly improved individual risk stratification for graft failure.
This graft injury phenotype was characterized by microvascular inflammation and complement split-product C4d deposition stratifying patients according to their complement binding capacity -not only to the presence or absence of DSAs- identified an additional group of patients with an increased risk of graft loss.
C1q-binding donor-specific anti-HLA antibodies remained strongly associated with the risk of graft loss regardless of the mean fluorescence intensity of the antibodies.
The presence of C1q and C4d do not provide the same prognostic utility. C1q testing may help identify patients at risk, despite C4d negativity.
Assessment for complement-binding donor-specific anti-HLA antibodies may provide an early indication of the potential for complement-mediated injury, without the functional requirement for progression through the pathway to C4d deposition
Heba Wagdy
3 years ago
HLA antibody evaluation CDCXM with antihuman globulin allowed detection of low level of antibodies. FCXM provide simultaneous and independent assessment of both T cell and B cell crossmatch. Flow PRA: A screening test, qualitatively detects the presence and/or absence of HLA antibodies. SAB assay: Semi-quantitative and detect and identify antibodies with higher specificity and sensitivity. Controversies in HLA antibody assessment:
Although MFI is reported as numerical value, It wasn’t intended to quantify antibodies and SAB assay is not approved as quantitative test.
MFI reflects fluorescence without reference to standard. Limitations:
There is no cutoff value at which organ transplantation is contraindicated or below it the transplant is considered risk free.
Intra-lab. variability of MFI results.
Reported MFI values are influenced by technologist dependent actions
MFI values may be falsely low due to interfering factors as complement split products and IgM HLA antibodies.
False positive reactivity may occur due to denatured antigens. New directions is to consider antibody reactivity to be epitope directed and not t individual HLA antigen Antibody pathogenicity:
The degree and strength of antibody binding may be influenced by antigen conformation which may be affected by differences in protein sequences.
The pathogenicity of antibodies can’t be determined according to MFI level. Functional methods to assess antibody pathogenicity:
Complement fixing ability of antibodies may be used to assess their pathogenicity, but results of studies were controversial regarding the association between complement fixing ability and poor graft outcome, some studies suggested that antigens can fix complement in vitro but may not do so in vivo and complement independent antibody mediated rejection may occur. Antibody assessment should be individualized according to each recipient.
Batool Butt
3 years ago
In this article the author have investigated the role of complement binding capacity of anti HLA antibodies in allograft rejection. Graft injury due to Anti HLA antibodies can be variable ranging from no damage to significant rejection. Complement fixing DSA which can be detected by C1q assay ,can cause both C4d positive ABMR and C4d negative ABMR .It is more important than non-complement fixing DSA, however, it is not correlated to intensity of DSA.
Current study enrolled 1016 patients from two transplantation centers in Paris between January 1, 2005, and January 1, 2011 , speculated C1q binding DSA (48%) was associated with severe ABMR than non-complement fixing antibodies(16%) and Graft survival was less in patients with C1q binding DSA ( 54%) than non C1q binding DSA ( 93%) . Other factors that worsened graft outcome included increment in donor age, each one minute increase in cold ischemia time, deceased donor and retransplant Therapeutic agents targeting complement (e.g., a C5 inhibitor [eculizumab] or a C1 inhibitor) are being used in transplant recipients
In conclusion, the presence of DSA is associated with poor allograft outcome and also helps in risk stratification of patients for graft loss .
Type of study: descriptive prospective cohort study with analytic component
Level of evidence: II
Sahar elkharraz
3 years ago
This article focus on role of complement binding Anti HLA Ab detection on graft survival.
It’s prospective cohort study done by Alexandre et al, in two transplant centers in Paris from first of January 2005 to first January 2011 with fallow up transplanted patients up to April 2012; all selected patients are ABO compatible & CDC T / B cells are negative. it’s a comparison study between 3 groups, patients had complement binding DSA anti HLA Ab and patients with non complement DSA anti HLA Ab and patients with out DSA HLA Ab. Those patients are screening clinical/ Functional/ histologically and immunological. They found that patients with complement binding DSA anti HLA b had quadruple risk of graft loss & AMR then patients with non complement binding DSA anti HLA Ab and non DSA Ab regardless MFI. they show evidence of micro vascular inflammation and deposits of Cd4 complement with graft capillaries. Anti HLA Ab are lymphotoxic and lead to activation of complement by binding of anti HLA to C1q. that’s why it’s important to screen for presence of C1q binding to DSA HLA Ab to improve outcome of graft. In this study clinical data collected prospectively at day0 / 6 month/ 1 year post transplant and then annually, also documented patients with acute rejection who developed deteriorated renal function/ proteinuria/ patients with histological evidence according to banff classification.
detection and characterization of DSA Ab: all patients tested for presence of circulating DSA in serum at Day 0 and at day of biopsy after one year of transplant or during attacks of acute rejection. circulating DSA for HLA A B Cw DR DQ Dp determined with use of single antigen flow bead on luminex platform. serum of C1q binding DSA anti HLA Ab with use of single antigen flow bead obtained.
Results of 3 groups of patients assessed according to presence or absence of DSA anti HLA Ab from total 1016 patients showed 700 with out circulating DSA/ 239 with non binding complement DSA anti HlA antibodies and 77 with complement binding DSA anti HLA antibodies. they show T cell mediated rejection & AMR and microvascular inflammation more in patients with DSA and C1q binding rather than patients with non C1q binding DSA anti HLA Ab. Also histological changes with more extensive micro vascular inflammation and transplant glomerulopathy and high score of peritubular capillary C4d deposits more in patients with C1q binding and DSA HLA Ab rather than other s; Also noticed decrease GFR level in those patients.
Kidney allograft survival rate are lower around 3.9 years in patients with C1q binding DSA in comparison with non specific C1q binding anti HLA Ab where those average survival rate 4.8 years. So patients with C1q binding DSA anti HLA antibodies are at risk of allograft rejection regardless of MFI high or low.
Eculizumab are C5 inhibitors or C1 inhibitors promise to use in kidney transplant to reduce risk of allograft rejection.
Q2: level 2
Q3: This study is prospective cohort study
Amit Sharma
3 years ago
I. Complement-Binding Anti-HLA Antibodies and Kidney-Allograft Survival
Please give a summary of this article
Anti-HLA antibodies are associated with graft injury. They have been shown to bind to C1q fraction of complement. A prospective cohort study was planned with the aim to identify the role of complement binding capacity of anti-HLA antibodies in renal graft prognosis.
The study group included a cohort of 1016 transplant recipients from 2 transplant centers in Paris between January 2005 and January 2011. Transplant recipients at another transplant center were taken as external validation cohort. All the patients were screened for donor-specific anti-HLA antibodies and their complement binding capacities at day 0 and 1 year post-transplant or at the time of acute clinical rejection. Kidney graft biopsy was done at 1 year post-transplant, or at the time of acute clinical rejection.
Out of 1016, 239 patients had non C1q binding DSA and 77 had C1q binding DSA. T cell mediated rejection was highest in C1q DSA group. C1q DSA group was associated with 3 times higher AMR than non C1q binding DSA group and had least GFR at 1 year among the 3 groups. The 5 year graft survival was least with the C1q DSA group (54% versus 93%^for non complement binding DSA and 94% for DSA negative patients).
The C1q DSA group was associated with higher and more severe rejection with microvascular inflammation, increased C4d positivity and increased graft loss. C1q DSA had strong association with graft loss without any relation to their MFI. So, presence of complement binding DSA can be taken as an early predictor for complement mediated injury progressing to AMR. Hence, these antibodies should be considered as a risk factor for AMR while assessing the prognosis of graft kidney.
What is the level of evidence demonstrated by this study?
Level II evidence
What this type of this study?
Prospective cohort study
amiri elaf
3 years ago
# Please give a summary of this article
* This study to investigate whether the complement binding capacity of anti-HLA antibodies plays a role in kidney allograft failure.
* In this study they enrolled patients who received kidney allografts at two transplantation centers in a population-based study.
* Patients were screened for the presence of circulating DSA and their complement binding capacity, Graft injury phenotype and the time to kidney allograft loss were assessed.
* The results is that
– The primary analysis included 1016 patients.
– Three distinct populations were identified after transplantation, according to the presence or absence of DSA
and complement-binding capacity:
– 700 patients without circulating DSA.
– 239 patients with non complement bindin DSA.
– 77 patients with complement-binding DSA.
# In the first year after transplantation, acute clinical rejection developed in 171 patients.
# 96 patients had T-cell mediated rejection (56%).
# 75 had antibody mediated rejection (44%).
* T-cell mediated rejection occurred in 14 of 77 patients with DSA plus C1q binding capacity (18%).
* In 30 of 239 patients with DSA but with out C1q binding capacity (13%).
* In 52 of 700 patients with out DSA(7%).
# Antibody-mediated rejection :
*Occurred in 37 patients with C1q binding DSA(48%).
* 38 patients with non C1q binding DSA(16).
# Among the patients with C1q-binding DSA :
* 67 had microvascular inflammation (87%).
* 28 had tubular and interstitial inflammation more severe abnormality (36%).
# Patients with C1q-binding DSA had more extensive microvascular inflammation , transplant glomerulopathy and high C4d deposition than both patients with non C1q binding DSA and patients with out DSA .
# Patients with C1q-DSA had a lower estimated glomerular filtration rate (GFR) at 1 year than patients with non C1q binding DSA and patients without DSA.
** After transplantation ( 5-year graft survival) :
# patients with DSA had significantly worse graft survival than patients without DSA , 83% vs. 94%; (P<0.001)
#. Patients with DSA were subsequently categorized according to complement binding capacity:
– Patients with C1q binding DSA had the poorest 5 year graft survival (54%), as compared with patients with nonC1q binding DSA.
# The risk of graft loss according DSA C1q status at day 0 and the status after transplantation revealed that they had the highest risk of graft loss .
# patients with C1q binding DSA had similar graft survival, regardless of whether the mean fluorescence intensity was
low or high.
# Conclusion:
– presence ofcomplement binding anti HLA donor specific antibodies after transplantation is strongly associated with graft injury and loss and that in corporation of this risk factor improves risk strati-
fication for graft loss
-The study gives a way for future therapeutic implications, agents targeting complement are increasingly being used in practice.
# What is the level of evidence demonstrated by this study?
LEVEL 2 evidence study.
# What this type of this study?
Prospective cohort study.
Huda Al-Taee
3 years ago
The road to HLA antibody evaluation:
The journey begins
Advances in techniques and technology paved the way to improved testing. These include adding antihuman globulin to DCD testing then the use of FCXM.
The game-changing moment in detecting HLA antibodies was the introduction of microparticle technology, including flow PRA & SAB assays. They are highly sensitive and specific, but these features have their challenges.
Controversies in HLA antibody assessment:
The primary concern about the result of SAB testing is that being a numerical value referred to as MFI. MFI reflects a given bead’s relative fluorescences without reference to a standard; many variables can affect this fluorescence.
Limitations:
MFI significant value is based on where and how thresholds are set to determine whether a bead is positive or negative.
MFI variability is compounded by vendor, antigen source, test reagents, and interlaboratory variability.
New directions:
From the mid-1960 to 1970, the term cross-reactive groups were used to describe the phenomenon of multiple unique HLA antigens that reacted with a single antibody.
Today, the terminology changed to epitopes, epletes. With this change, what was previously known as third-party antibodies are now considered DSA.
Conformation- an integral component to antibody pathogenicity
The degree of antibody binding is influenced by antigen conformation, affecting the MFI value.
Antigens versus antibodies:
DSA that binds a bead in vitro may fail to attach to the corresponding antigen in vivo because its expression is too low. In vivo, a lower level of HLA antigen expression compared with beads could provide a target to which the DSA can bind. Therefore, the pathogenicity of an antibody cannot and should not be assumed strictly on MFI levels.
Functional methods to assess antibody pathogenicity:
Most of the studies focused on two things: complement-fixing and IgG subclass.
The largest study evaluating complement-fixing DSA with poor graft survival found a clinical significance only post-transplantation.
Studies found that not all sera containing potent complement-fixing antibodies can fix complement in vitro; the explanations for this:
the presence of complement-fixing molecules such as heparin sulfate and/ or increased level of the decay-accelerating factor.
hexamerization of IgG Fc fragments is required to initiate the activation of the complement cascade.
Guides to antibody assessment
Challenging cases need a multidisciplinary team to outline the immunological complexities of particular donor-recipient pair and to discuss issues that typically one or more of the participants do not consider.
So the road toward HLA antibody detection and identification is still under construction.
Sherif Yusuf
3 years ago
The Road to HLA Antibody Evaluation: Do Not Rely on MFI
HLA antibodies detection Journey:
– Starting by CDC XM and because of lower sensitivity and specificity, human anti- goblin was added to increase the sensitivity (Anti-Human Globulin (AHG) augmented CDC), followed by FCM XM which provide good sensitivity an specificity when compared with CDC and AHG- augmented CDC and lastly DSA detection using luminex technique which is highly sensitive and specific technique for detection of HLA antibodies either donor on non-donor specific
Significance of DSA MFI level
– Luminex gives a quantitative assessment of the strength of DSA through measurement of MFI, the significance of MFI and its correlation to graft outcome is debatable which make the setting of certain threshold for significant MFI is extremely difficult, this may be explained by the following facts :
1- Different thresholds for detection of DSAs were set with variable MFI cutoffs so false negative and positive results can occur due to this variation, this means a positive result in one center is negative in another (interlaboratory variability).
2- Intra – laboratory variability due to reagents also affects the result of DSA MFI
3- The DSA MFI doesn’t correlate well with XM, It may predict negative XM (if it is below threshold) but not positive ones (have negative predictive value). Thus some patients may have high MFI and negative XM
4- Significance may differ according to HLA antigen to which antibody is directed, so significant MFI for DSA directed to HLA class I may differ from HLA directed to class II
5- The MFI doesn’t correlate well with graft outcome, some patients may have DSA with high MFI (> 10000) and have acceptable graft survival, others may have very low DSA (MFI 100) and develop graft loss
6- DSA response to treatment indicated by decreasing MFI may be associated with better graft survival
7- Complement fixing AB (determined by the C1q assay) are proved by some studies but not all to be more clinically significant than non-complement fixing AB , other studies reported this significance in denovo DSA and not performed DS, also complement-fixing ability is not correlated well with the intensity of DSA, this means that there may be a DSA with high intensity, but is not complement-fixing.
8- Expression of HLA antigen on kidney tissue may affect the significance of DSA MFI, for example if DSA directed to antigen with a low expression on kidney tissue may result in high MFI invitro, while invivo it may be either nonsignificant in some cases but in others become significant due to increased expression of this antigen invivo.
9- False positive result can occur due to :
• Denatured antigens attached to beads, leading to alteration in protein configuration
• Sharing of public epitopes among several antigens (HLA molecule-based matching). Epitope matching which includes the use of epitopes as the focus in HLA typing (epitope-based matching) eliminate this false-positive results this is called the highly specific SAB system
10- False-negative results can occur due to :
• High levels of IgM that can bind to antigen beads and prevent binding to actual IgG-DSA, this is called prozone efect or hook effect
• Very high level of DSA that agglutinate so fail to bind to the beed,
• Appearance of antigenic epitope on multiple beads, thus DSA bind to multiple beads thus diluted giving false-negative result, this will be detected by SAB.
Innocent lule segamwenge
3 years ago
Complement-binding anti-HLA antibodies and kidney –allograft survival
Introduction
It is well established that anti-HLA antibody production leads to kidney allograft failure.
The clinical outcomes of these antibodies range from causing no damage to severe allograft injury.
This varied difference in injury caused by these antibodies led the authors to postulate that this could be related to the difference in complement binding capacity of these antibodies.
C4d deposition in glomerular capillaries is considered to be the biological footprint of ABMR in the allograft.
Binding of antibodies to C1q is the first and perhaps most important step in the activation of the classical complement pathway.
Objectives
To define the complement binding capacity of anti-HLA antibodies.
To define patterns of allograft injury according to the C1q binding capacity of ant-HLA DSA
To determine utility of C1q binding DSA in risk stratification of kidney-allograft loss.
Methods
Prospective cohort study from January 2005 to January 2011
Conducted in France at two hospitals (Necker and Saint-Louis Hospitals)
Study population: All ABO compatible, and CDC T and B negative patients who underwent kidney transplantation during the study period
Clinical data was collected from the national registries.
Acute clinical rejection was defined by; Deterioration in graft function, proteinuria, and histological evidence of rejection according to Banff criteria.
Patients were treated with similar immunosuppression and rejection protocols
Biopsy results from 1 year protocol biopsies and for cause biopsies were used. C4d staining was done on all biopsies.
All patients were tested for anti-HLA DSA on pre-transplant stored blood samples and at the time of biopsy.
Results
1016 patients were included in the analysis.
700/1016 no DSA
239/1016 non-complement binding DSA
77/1016 complement (c1q) binding DSA
Kidney allograft injury
171/1016 had acute clinical rejection
96/171 (56%) had TCMR, 75/171 had ABMR
14/77 had TCMR + C1q binding DSA
30/239 had TCMR + non-C1q binding DSA
52/700 had TCMR + no DSA
37/77 had ABMR + C1q binding DSA
38/239 had ABMR + non-C1q binding DSA
Patients with C1q binding DSA were more likely to have;
✓ Extensive Microvascular inflammation
✓ Severe score for tubular and interstitial inflammation
✓ Interstitial fibrosis and tubular atrophy
✓ Severe transplant glomerulopathy
Allograft survival
Patients with DSA had worse graft survival than those without.
Patients with C1q binding DSA had the worst allograft survival.
Predictors of graft loss;
1. Low 1 year GFR
2. Interstitial fibrosis and tubular atrophy
3. Transplant glomerulopathy
4. C1q binding DSA
Conclusion
This has study has convincingly demonstrated the deleterious effects of DSA in terms of causing worse graft inflammation and loss of function, but most importantly the worst effects of DSA are seen with the C1q binding DSA.
Mohamed Saad
3 years ago
The Road to HLA Antibody Evaluation: Do Not Rely on MFI.
Introduction:
Positive CDC cross match was a contraindication for Transplantation but some patient got good graft survival and vice versa some patients with negative CDC cross match had many attacks of rejections with poor graft outcome so we are looking for another reliable method to detect clinical relevant of HLA antibody. The Journey Begins:
To improve CDC sensitivity ,AHG added to detect lower level of HLA antibody then flow-cytometry technique started with high sensitivity and with independent assessment of both T &B Cell cross match. Now SAB assay provided with high sensitivity and specificity in detection and identification of HLA class I&II. Controversies in HLA Antibody Assessment:
MFI is a numerical value of SAB result which reflects a given bead’s relative fluorescence without reference to a standard.
Which used to assess antibody strength and to correlate number with clinical outcomes especially after treatment of Rejection.(which is still debatable). Be Prepared to Stop: Limitations:
MFI cutoff values are different from one center to another and also according o transplantation program, Many studies reveled that low MFI associated with poor graft outcome and vice versa, and many laboratory factors affected on the result of MFI and keep it dynamic more and more and difficult to relay on it to interprets with clinical practice. Rerouting: New Directions:
Some antibody react with more than antigens which share together(which mainly epitope that share antigenicity in more than HLA antigen) and so we should change antigens bead to epitopes. Bumps in the Road: Conformation—An Integral Component to Antibody Pathogenicity?
The degree and strength of antibody binding is influenced by antigen conformation , e.g. an antibody to Bw6 was identified on class I SAB with MFI values >17 000 except for the B*46:01- and B*73:01-bearing beads, which had MFI values of ~2000 and ~1500, respectively, which might be depend on sequence of amino acids(eplet) which different from site in epitopes to another which change antigenicity.
The difference at this position infers a change in epitope conformation that diminishes antibody affinity for the target antigen , all these can affect MFI value. Divided Highway: Antigens Versus Antibodies:
Antigens might be expressed in vivo more than invitro which induced antibody response so DSA with low MFI might be not reflect the antigen expression in vivo so its difficult to relay on MFI for clinical outcome. Merge: “Functional” Methods to Assess Antibody Pathogenicity:
Complement fixing characteristics of HLA antibodies, by adding (c1q, c3d or c4d) as a surrogate for complement system in vivo many studies revealed its association with poor graft outcome and other not .but sometimes ABMR occurred due to non-complement dependent pathway. Guides to Antibody Assessment:
So antibody assessment needs more and more technical advances and innovations , but still we should work as a multi -disciplinary team and deal case by case assessment to guide antibody with each case.
To deal with the total view clinical and technical aspects.
Tahani Ashmaig
3 years ago
☆Complement-Binding Anti-HLA Antibodies
and Kidney-Allograft Survival
_____________________________________________
Summary:
▪︎In this study the authors hypothesized that the complement binding properties of donor-specific anti-HLA antibodies detected after transplantation are involved in kidney-allograft failure. So, they conducted this study to:
1) Define the full spectrum of kidney-allograft injury according to the C1q-binding properties of donor-specific anti-HLA antibodies in a large population-based study.
2) Determine whether assessment for the presence of C1q-binding donor-specific anti-HLA antibodies after transplantation might improve risk stratification for kidney-allograft loss.
Methods:
__________
▪︎ Study population : Patients who received kidney allografts at two transplantation centers in Paris between January 1, 2005, and January 1, 2011. Patients were followed until April 15, 2012. An external-validation cohort was also included.
▪︎All transplants were compatible with the ABO blood group.
▪︎ A negative result of cross-matching for IgG T-cell and B-cell complement-dependent cytotoxicity was required for all recipients.
Clinical Data:
______________
▪︎ Anonymized data from registries were entered on day 0 and 6 months and 1 year after transplantation for each patient and were updated annually thereafter.
▪︎ The derivation-cohort data and validation-cohort data were obtained.
▪︎All cases of acute clinical rejection (defined by deterioration in graft function, proteinuria, or impaired renal function and histopathological evidence of rejection, according to the consensus rules of the international Banff classification criteria), were documented.
◇Histologic and Immunochemical Tests:
____________________________________________
▪︎ Biopsies were performed in patients with/without acute clinical rejection episodes diagnosed in the first year of transplantation and were scored and graded from 0 to 3 according to the updated Banff criteria with respect to the presence of donor-specific anti-HLA antibodies, C1q-binding status, and clinical course.
▪︎ C4d staining was performed
◇Detection and Characterization of Donor-Specific Antibodies:
______________________
▪︎All patients were tested for the presence of circulating donor-specific anti-HLA on Luminex platform.
▪︎ Serum samples from patients with circulating donor-specific anti-HLA antibodies were analyzed for the presence of C1q-binding donor-specific anti-HLA antibodies with the use of single-antigen
flow bead assays.
♧ Discussion:
_______________
▪︎The presence of complement-binding donor-specific anti-HLA antibodies detected in the first year after transplantation was an independent predictor of kidney-allograft loss more than 5 years after transplantation and significantly improved individual risk stratification for graft failure.
▪︎Patients with complement-binding donor-specific anti-HLA antibodies after transplantation had a graft injury phenotype characterized by microvascular inflammation and complement split-product C4d deposition.
▪︎ C1q-binding donor-specific anti-HLA antibodies remained strongly associated with the risk of graft loss regardless of the mean fluorescence intensity of the antibodies.
▪︎ C1q testing may help identify patients at risk, despite C4d negativity.
▪︎Assessment for complement-binding donor-specific anti-HLA antibodies may provide an early indication of the potential for complement-mediated injury, without the functional requirement for progression through the pathway to C4d deposition.
▪︎ Detection of complement-binding donor-specific anti-HLA antibodies may indicate which of the antibodies present have the capacity to activate the complement cascade.
☆This study may provide a basis for future clinical trials (e.g., a C5 inhibitor [eculizumab] or a C1 inhibitor)
☆The level of evidence of this study:
____________________________________
Level II
☆The type of this study:
________________________
Population based large cohort study
Last edited 3 years ago by Tahani Ashmaig
MICHAEL Farag
3 years ago
What this type of this study?
This is a cohort study What is the level of evidence demonstrated by this study?
Level 2 of evidence.
Summary of the article Complement-Binding Anti-HLA Antibodies and Kidney-Allograft Survival This article discusses the impact of the anti HLA antibodies on the kidney graft survival. Although anti-HLA antibodies are considered to be harmful, there is a wide spectrum of graft injury related to these antibodies, ranging from no recognizable damage to florid rejection.11,12 Such a varied effect underscores the need to define distinct graft phenotypes and outcomes according to the presence or absence and characteristics of donor-specific anti-HLA antibodies after transplantation. Methods We enrolled all consecutive patients who underwent kidney transplantation at Necker Hospital and Saint-Louis Hospital (Paris) between January 1, 2005, and January 1, 2011, in this populationbased study. Patients were followed until April 15, 2012. We also included an external-validation cohort comprising patients who underwent kidney transplantation at Foch Hospital (Suresnes, France) between January 1, 2004, and January 31, 2010 The transplantation allocation system was identical for the three centers and followed the rules of the French national agency for organ procurement (Agence de la Biomédecine) All transplants were compatible with the ABO blood group.A negative result of cross-matching for IgG T-cell and B-cell complement-dependent cytotoxicity was required for all recipients Clinical Data Clinical data on the donors and recipients in the derivation cohort and the validation cohort were obtained from two national registries. Anonymized data from these registries are prospectively entered at specific time points for each patient (on day 0 and 6 months and 1 year after transplantation) and are updated annually thereafter) The derivation-cohort data were obtained from the database on April 15, 2012, whereas the validation-cohort data were obtained on December 19, 2012. We documented all cases of acute clinical rejection, defined by deterioration in graft function, proteinuria, or impaired function and histopathological evidence of rejection, according to the consensus rules of the international Banff classification criteria. Immunosuppression protocols and treatment of allograft-rejection episodes after transplantation were similar among the centers. Histologic and Immunochemical Tests We used specimens from protocol-specified graft biopsies performed 1 year after transplantation in 845 patients without any acute clinical rejection episodes diagnosed in the first year after transplantation, as well as specimens from biopsies performed in 171 patients with acute allograft rejection during the first year after transplantation. All graft-biopsy specimens were scored and graded from 0 to 3 according to the updated Banff criteria by three trained pathologists who were unaware of the patient’s status with respect to the presence of donor-specific anti-HLA antibodies, C1q-binding status, and clinical course. C4d staining was performed by means of immunochemical analysis on paraffin sections with the use of polyclonal human anti-C4d antibodies. Detection and Characterization of Donor-Specific Antibodies All patients were tested for the presence of circulating donor-specific anti-HLA antibodies in banked serum samples. donor-specific anti-HLA antibodies were analyzed in a blinded fashion. Statistical Analysis We used means and standard deviations for the description of continuous variables, with the exception of mean fluorescence intensity, for which we used the mean and standard error. We compared means and proportions using Student’s t-test and the chi-square test. Survival was analyzed from the time of transplantation to a maximum of 7 years, with kidney-graft loss as the event of interest Results Baseline Characteristics of the KidneyAllograft Recipients In total, 1016 patients undergoing renal transplantation (695 at Necker Hospital and 321 at SaintLouis Hospital) were included in the main analysis. Three distinct populations were identified after transplantation, according to the presence or absence of donor-specific anti-HLA antibodies and complement-binding capacity. – 700 patients without circulating donor-specific anti-HLA antibodies – 239 patients with non–complement-binding donor-specific anti-HLA antibodies, – 77 patients with complement-binding donor-specific anti-HLA antibodies Kidney-Allograft Injury In the first year after transplantation, acute clinical rejection developed in 171 patients: – 96 patients had T-cell–mediated rejection (56%) – 75 had antibody-mediated rejection (44%). T-cell– mediated rejection occurred in – 14 of 77 patients with donor-specific anti-HLA antibodies plus C1q-binding capacity (18%), – in 30 of 239 patients with donor-specific anti-HLA antibodies without C1q-binding capacity (13%) – in 52 of 700 patients without donor-specific anti-HLA antibodies (7%) Antibody-mediated rejection occurred in – 37 patients with C1q-binding donorspecific anti-HLA antibodies (48%) – 38 patients with non–C1q-binding donor-specific anti-HLA antibodies (16%) Kidney-Allograft Survival The median follow-up after transplantation was 4.8 years. The risk of graft loss according to the donor-specific anti-HLA antibodies–C1q status at day 0 and the status after transplantation revealed that patients with C1qbinding donor-specific anti-HLA antibodies after transplantation had the highest risk of graft loss.
Doaa Elwasly
3 years ago
The HLA (second paper)
Searching for a new method to Identify clinically relevant HLA antibodies of clinical importance was a challenging
The Flow PRA is a screening test to detect HLA antibodies qualitively
while SAB assays is a semiquantitative test detecting the antibody specificity.
These two tests are sensitive and specific .
Luminex was specific and sensitive in detecting HLA antibodies.
mean fluorescence intensity (MFI) is a numerical value reported from SAB results .
It reflects a certain bead’s relative fluorescence which is affected by many variables.
The drawbacks
-If the threshold is low, false-positives may prevent a patient from receiving a compatible graft ;and if high, there may be increased risk that a patient will have AMR.
-Most studies demonstrated that low MFI levels correlated with favorable outcome and high indicated poor graft outcome but the range of MFI values is not standardized.
-Also SAB results are variable between different labs
-For MFI values stems from interferences due to antibody-dependent complement activation. Complement split products bind to antigen–antibody complexes on the beads blocking binding sites for secondary antibodies
-Low MFI DSAs associated with poor outcomes can be explained by competing of IgM HLA antibodies, interfering with the binding of IgG HLA antibodies
– False-positive result can occur due to antibody binding to denatured antigens.
-On the other hand high-MFI DSAs were not associated with poor graft outcome this can be attributed to denatured proteins .
– It is unknown if the reactivity to native and denatured HLA antigens is hazardous or not
Cross-reactive groups (CREGs) described the serologic phenomenon of multiple specific HLA antigens that reacted with a single antibody.
Therefore the antibody reactivity is directed to the epitope rather than to individual HLA antigens, known before as non-DSAs, now considered as DSAs.
The degree of antibody binding is affected by antigen formation.
The current version of HLAMatch-maker and the epitope registry factor (eplets) that have recently become a major component of compatibility determination in many laboratories.
Difference in MFI may be due to effect of more than one of the amino acid differences.
DSA that binds a bead in vitro may not bind to the corresponding antigen in vivo due to low expression .
On the contrary Wiebe et al, stated that the de novo antibody produced post transplantation are directed to HLA-DQ antigens despite low expression of the DQ loci .
The harmful effect of an antibody cannot not be assessed based solely on MFI levels.
Some studies demonstrated significant correlation between complement fixation and poor allograft outcome while others did not.
Meanwhile complement-independent AMR can occur .
HLA antibody detection and identification is subjected to further research
Drtalib Salman
3 years ago
DSA carry negative impact on graft survival but need to know more about DSA MFI, anti HLA class 1,antiHLA class 2,anti A,B,DR, DQDP, IG g subclass ,preformed or post transplant
IN THIS ARTICLE AIM OF STUDY TO COMPARE CPLEMENT FIXING FROM NON COMLEMENT FIXING DSA ON allograft survival .
1016 patient enrolled in this study from NECKER HOSPITAL and Saint -LOUIS HOSPITAL (PARIS) with external validation from FOCH-HOSPITAL
Period from 2005 to 2011.
845 underwent protocol biopsy 1 year after transplantation .
171 for cause biopsy (graft rejection).
DSA measured at 0 and at one year post transplantation .
C1Q binding status was assessed.
5 year graft survival in patient with complement fixing DSA was inferior to those who has non complement fixing DSA .
Risk stratification of patient change if we know that DSA complement fixing from non complement fixing but from
my point of view apart from risk assessment and prognostic information need to know if we need to do C1Q status to All DSA positive patient ,
and if there is any change in desensitizing regimen or line of management of Ab mediated rejection between C1Q positive or negative patient ?,
what about cost effectiveness?.
What this type of this study?
This is cohort study
What is the level of evidence demonstrated by this study?
Level 2 of evidence .
Heba Wagdy
3 years ago
The effect of anti-HLA antibodies post transplant varies from unrecognized damage to florid rejection according to the characteristics of anti-HLA antibodies.
The capacity of anti-HLA antibodies to bind complement and activate the classic complement cascade determines the cytotoxicity of these antibodies.
This study aimed to determine whether the assessment of presence of C1q binding DSA will improve risk stratification for graft loss.
A prospective cohort study included 1016 kidney transplant recipients with compatible ABO blood group and negative CDC crossmatch, they were followed up for 4.8 years. It evaluated the immunologic characteristics before and after transplantation including graft phenotype.
Acute rejection was defined by deterioration of graft function, proteinuria and histopathological evidence of rejection.
Patients were tested for DSA at time of transplantation, one year post transplant and during episode of acute rejection.
Sera of patients with DSA were analyzed for presence of C1q binding DSAs.
Patients were identified according to absence or presence of DSA and complement binding capacity.
Detection of complement binding DSA in first year post transplant was an independent predictor of graft loss 5 years post transplant and significantly improved risk stratification for graft failure.
Patients with complement binding DSA had graft injury characterized by microvascular inflammation and C4d deposition.
C1q binding DSA was strongly associated with the risk of graft loss regardless the MFI of antibodies.
Assessment for complement binding DSA may indicate the potential of complement mediated injury before progression through the pathway to C4d deposition.
Identifying complement binding ability may be important as new therapeutic agents as C5 inhibitor (eculizumab) and C1 inhibitor targeting complement are increasingly used in kidney transplant recipients. Limitations:
It didn’t investigate the changes in capacity of DSA to bind complement and the effect of treatment on these antibodies.
prospective cohort study
Level of evidence II
Filipe Prohaska Batista
3 years ago
Please give a summary of this article
Two transplant centers in Paris were evaluated over a period of six years, which monitored the circulation of HLA antibodies and complement-binding capacity after transplantation and its role in graft failure.
All patients had ABO compatibility and negative results for both crossmatch and complement-dependent cytotoxicity. SAB Luminex collected on day 0 and 365 dosing HLA A, B, Cw, DR, DQ, DP and C1q binding associated with biopsies with evidence of clinical and laboratory rejection or after a period of one year.
1016 patients were selected, 845 without symptoms and 171 with TCMR (56%) and AMR (44%).
When stratifying the data, the presence of C1q+ showed a higher risk of losing the graft and showing lower levels of Creatinine Clearence. Histopathological findings showed microvascular infiltration (87%), interstitial tubular inflammation (36%), endarteritis (23% and transplant glomerulopathy (22%).
The presence of c1q complement binding after transplantation showed a Harzard ratio of 4.48 independent of the positivity of other DSAs.
The presence of C1q+ is an independent factor of graft loss and independent of MFI values, only confirming the need to expand the investigation of ASD beyond HLA standards, in addition to more sensitive and specific methods.
What is the level of evidence demonstrated by this study? What this type of this study?
Cohort study – IIb
Wael Jebur
3 years ago
Prospective population based study carried out in France between 2005 through 2012.
Aim: To assess the correlation of complement binding DSAs with allograft survival and post transplantation complications in comparison to patients with non complement binding DSAs and patients with negative DSAs.
Method:
1016 patients were enrolled in this prospective study.HLA A,B,Cw and DR,DQ and DP were assessed by Luminex
All the patients were tested for DSAs at the time of transplantation and one year post transplantation.
Protocol allograft biopsy was performed one year post transplantation or at any time during the first year if any patient developed acute rejection or allograft dysfunction,looking for changes related to rejection process according to Banff classification.
C1q binding test was performed in all patients with circulating DSAs to categorize into complement fixing and non complement fixing DSAs.
Results:
3 distinct populations identifed :
700 patients with negative DSAs.
239 patients with DSAs non complement fixing.
77 patients found to have complement fixing DSAs.
171 patients developed acute rejection in the first year post transplantation, of which 96 had CMR and 75 had AMR.
Of the 96 patients with CMR :
14 with C1q positive DSAs patients.
30 C1q negative DSA
52 DSA negative patients.
Of the 75 patients with AMR:
37 patient with C1q positive DSAs
38 patients with C1q negative DSAs.
Of the 77 patients with c1q positive DSAs 37 (48%)developed AMR.
Of the 239 patients with c1q negative DSAs 38(16%) developed AMR.
This result revealed strong evidence that c1q positive DSAs are associated with higher incidence of AMR in comparison to C1q negative DSAs patients and those patients with negative DSAs(48% vs16%) statistically significant with p value of less than 0.001.
Further more ,c1q positive DSAs patients showed more extensive micro vascular inflammation and transplant glomerulopathy and higher scores for graft peritubular c4d deposition than for both C1q negative DSAs and negative DSAs patients.stastically significant.
Gfr of the c1q patient was significantly lesser than c1q negative DSAs and patients with negative DSAs over median follow up period of 4.8 years.
Similarly 5 years graft survival was 54% in c1q positive DSAs patients vs 93% and 99 % for those with c1q negative DSAs and DSAs negative patients respectively.statistically significant with P value of less than0.001.
An important observation was made by this study, that is MFI level, as it was non significant risk factor in those patients who are c1q positive DSAs (same risk in those with MFI above or below 6000)
Conclusion:
Complement fixing DSAs are associated with higher risk of AMR and bad long term outcome.
It’s representing an important suttogate marker for evaluation and risk stratification for kidney transplant recipients.
Anti-HLA antibodies is important for successful transplantation, and activation of the complement cascade is involved in antibody-mediated rejection.The analysis enrolled 1016 patients. The graft survival was 54% in patient with mplement-binding
donor-specific anti-HLA antibodies of as compared with patients with non–complement-binding donor-specific anti-HLA antibodies (93%) and patients without donor-specific anti- HLA antibodies (94%). The presence of comple-
ment-binding donor-specific anti-HLA antibodies after transplantation was associated with high a risk of graft loss . AMR , sever microvascular inflammation and C4D deposition is more and extensive in complement binding antibodies .
C1q assay may lead to distinguishing patients at risk, Although C4d is specific ,it is less sensitive as an indicator of antibodies rejection. The presence of complement-binding anti-HLA donor specific antibodies after transplantation is strongly associated with graft injury and loss
· What is the level of evidence demonstrated by this study? Level 11
· What this type of this study?Cohort prospective study
.
Complement-Binding Anti-HLA Antibodies and Kidney-Allograft Survival
This is a prospective cohort study with level II evidence involving kidney recipients at two transplantation centers in Paris between January 1, 2005, and January 1, 2011. The objective is to assess the association of complement-Binding Anti-HLA Antibodies and Kidney-Allograft Survival. A total of 1016 kidney transplant recipients recruited of which 700 patients without circulating donor-specific anti-HLA antibodies, 239 patients with non–complement-binding donor-specific anti-HLA antibodies, and 77 patients with complement-binding donor-specific anti-HLA antibodies.
Following renal transplant in the first year, a total 171 kidney transplant recipients developed acute rejection of which 96 patients; 56% had TCMR and 75 patients; 44% had AMR. TCMR is relatively higher among renal transplant recipients with donor specific anti HLA antibodies and C1q binding capacity. The median follow-up duration was 4.8 years. Those with donor specific anti HLA antibodies had poor renal graft survival especially those with C1q binding capacity donor specific DSA had poor 5-year graft survival.
The independent predictors of graft loss include low estimated GFR at 1-year, interstitial fibrosis and tubular atrophy, glomerular and peritubular inflammation, transplant glomerulopathy and the
presence of complement-binding donor specific anti-HLA antibodies.
In conclusion, complement-binding capacity of donor-specific anti-HLA antibodies allow early recognition of patients at high risk of allograft rejection and prompt action to be taken.
Complement-Binding Anti-HLA Antibodies and Kidney-Allograft Survival
Since the presence ofAnti-HLA antibodies is one of the main barriers for successful transplantation, and activation of the complement cascade is involved in antibody-mediated rejection. The aim of this paper to assess whether the complement-binding capacity of anti-HLA antibodies plays a role in kidney-allograft failure.
-Study type: prospective cohort study with level II evidence.
Methods:
Study included patients received kidney allografts in 2 centres in Paris between January 1, 2005, and January 1, 2011, in a population-based study. Patients were screened for the presence of circulating DSA antibodies and their complement-binding capacity. Rejection phenotype and the time to kidney allograft loss were assessed.
Results:
The primary analysis included 1016 patients. Patients with complement-binding donor-specific anti-HLA antibodies after transplantation had the lowest 5-year rate of graft survival (54%), as compared with patients with non–complement-binding donor-specific anti-HLA antibodies (93%) and patients without donor-specific anti HLA antibodies (94%). The presence of complement binding donor-specific anti-HLA antibodies after transplantation was associated with a risk of graft loss that was more than quadrupled when adjusted for clinical, functional, histologic, and immunologic factors. These antibodies were also associated with an
increased rate of antibody-mediated rejection, a more severe graft injury phenotype with more extensive microvascular inflammation, and increased deposition of C4d within graft capillaries. Adding complement-binding donor specific anti-HLA antibodies to a traditional risk model improved the stratification of patients at risk for graft failure.
There are therapeutic implications of knowing the pathways responsible for kidney allograft loss. Since promising therapeutic agents targeting complement (e.g., a C5 inhibitor [eculizumab] or a C1 inhibitor) are increasingly used in patients undergoing transplantation and ABMR.
One limitation of this study was that it was not designed to provide kinetics of the capacity of anti-HLA antibodies to bind complement
or the effect of treatment on these antibodies.
Summary
Anti-HLA antibodies can impact transplant outcome. One of the methods through which they act is by binding to complement, activating the complement cascade.
Patients with complement binding donor specific anti HLA antibodies appear to have very low rates of graft survival in comparison with recipients who have anti HLA antibodies that are not complement binding. Graft loss risk is more than 4 times. Other risk associations include AMR, and increased deposition of complement fraction C4d in graft capillaries.
Thus, complement binding anti HLA antibody assessment can help in identifying recipients who are at high risk of graft loss. Both C1q and C4d assessment are to be done. Even if C4d is negative, C1q has to be done because C4d lacks sensitivity although it is specific.
In conclusion, this study finds that post transplant presence of complement binding anti HLA DSA is strongly correlated with high risk of graft injury and graft loss. It is recommended to use this fact for risk assessment. C1q assessment is a significant tool in this aspect.
Level of evidence
Level of evidence is 2.
Type of study
Prospective cohort study
Dear all thankyou for participation but l noticed some new names apart from the regular active ones.
This is fine but l would like to see contributions in the scenarios.
Please be encouraged to take part no problem to make mistakes that is how we all learned.
early studies recognized that ant HLA Ab are destructive .
-Then recognition that complement system activation is a key component of ABMR .
– recognition of c4d deposition to be a footprint of ABMR .
-The capacity of anti-HLA antibodies to bind C1q, may determines the cytotoxic potential of these antibodies,
-Patients with C1q-binding DSA had a lower estimated (GFR) than did patients with non–C1q-binding DSA and patients without donor-specific anti-HLA antibodies .
– Patients with DSA had significantly worse graft survival than patients without DSA .
– patients with C1q binding DSA had the poorest graft survival after transplantation as compared with patients with non– C1q-binding DSA and patients without DSA .
-patients with C1q binding donor-specific anti-HLA antibodies after transplantation had the highest risk of graft loss .
– the presence of complement-binding DSA detected in the first year after transplantation was an independent predictor of kidney-allograft loss after trans plantation and significantly improved individual risk stratification for graft failure.
-Patients with complement-binding DSA had a graft injury phenotype characterized by microvascular inflamation and complement split-product C4d deposition.
– Although the MFI of DSA is widely used in clinical practice for risk stratification, THIS study
showed that C1q-binding DSA remained strongly associated with the risk of graft loss regardless of the MFI
of the antibodies.
– From a prognostic perspective, this study provide support for the finding that C1q and C4d do not provide equivalent predictive information. C1q testing may help identify patients at risk, despite C4d negativity.
– this study found that C4d, although specific, lacks sensitivity as an indicator of humoral rejection.
– the presence of complement-binding DSA after transplantation is strongly associated with graft injury and loss and that in corporation of this risk factor improves risk stratification for graft loss.
TYPE OF STUDY
prospective cohort study
LEVEL
level II
descriptive prospective cohort study
Level of evidence: II
anti-HLA antibodies are lymphocytotoxic,13 activation of the complement cascade has been considered to be a key component of antibody-mediated allograft rejection, and C4d deposition in renal capillaries has been considered the footprint of antibody-mediated allograft damage.
Plus complement fraction C1q, the first step in activation of the classic complement cascade.
Complement-binding donor-specific anti-HLA antibodies remained independently associated with the risk of kidney-allograft loss after adjustment for the mean fluorescence intensity of donorspecific anti-HLA antibodies
C1q-binding donor-specific anti-HLA antibodies remained strongly associated with the risk of graft loss regardless of the mean fluorescence intensity of the antibodies
may have implications for other transplanted organs such as the heart, lungs, and small bowel,
C1q testing may help identify patients at risk, despite C4d negativity
C4d, although specific, lacks sensitivity as an indicator of humoral rejection
promising therapeutic agents targeting complement (e.g., a C5 inhibitor [eculizumab] or a C1 inhibitor)
Good effort.
Please give a summary of this article
What is the level of evidence demonstrated by this study?
What this type of this study?
prospective cohort study with level 11 a of evidence
NEJM with high impact factor 91.24 in 2022
Introduction:
Kidney transplant survival and outcome compromised overtime due to the risk of antibody mediated rejection; anti- HLA antibodies considered the crucial part in the classic complement pathway activation resulted in antigen -antibodies alloimmune response with C4split product deposition which is the the foot print of alloimmune response. the study aims to assess the role of HLA Abs complement activation and the renal transplant graft failure
Methodology:
Population based prospective cohort study from two transplant centers in Paris, with external validation from cohort from third center sharing the same local registry database 2, they included 1016 patients with anti-HLA abs positive kidney transplant from the period between -2005-2011 and follow up at 0 time, 6, 12 months then annual review, last follow up extended till April 2012 with final outcome of graft survival. All included cases with ABO compatible transplantation with negative crossmatch for both T and B cells with similar allocation criteria from the three centers, the baseline characteristics shows in table 1, Patients with previous sensitization with DSA +ve pre transplants have more C1q complement activation compared to those with DSA -ve pre transplantation. They divided them into three groups according to the presence of complement binding anti HLA Abs, non-HLA DSA, non-complement binding DSA
Acute rejection defined as acute clinical renal function deterioration with proteinuria and histological diagnosis of rejection by protocol biopsies at one year post transplantation, in majority 845 cases while another 171 cases underwent graft biopsy as per clinical indication with acute graft dysfunction all biopsies reviewed based on updated Banff diagnostic score 0-3 by three independent pathologists and use IHC staining for C4D detection.
All have similar immunosuppression therapy for treatment of AR
Results:
Patients with C1q binding DSA have higher rate of AMR 48% and more sever microvascular injury with intimal arteritis , tubular and interstitial inflammation score of >2 , Tg , and higher score for C4D staining associated with lower GFR upon 1 year FU with progressive graft dysfunction as compared with non-complement binding DSA .Median FU 4.8 Years ( range 0.2-7) after transplantion and median FU for C1q binding DSA was 3.9 years
Renal survival was worse In those with anti HLA DSA , 5 year graft survival was 88% and even more worse graft survival was reported in those with anti HLA C1 q binding DSA( 54%), and also associated with higher rate of graft failure .
Excellent well understood and analysed.
The Relevance of Complement C4d Staining in Renal Allograft
Biopsies:
Abstract:
The complement binding properties of donor-specific anti-HLA
antibodies detected after transplantation are involved in kidney-
allograft failure.
Activation of the complement cascade has been considered to be a
key component of antibody-mediated allograft rejection, and C4d
deposition in renal capillaries has been considered the footprint of
antibody-mediated allograft damage.
The capacity of anti-HLA antibodies to bind complement fraction C1q,
which is the first step in activation of the classic complement
cascade, determines the cytotoxic potential of these antibodies, and
an assessment of their complement binding capacity may be useful
both for risk stratification and for diagnosis of antibody-mediated
rejection.
Methods:
Study Population:
All consecutive patients who underwent kidney transplantation at
Necker Hospital and Saint-Louis Hospital (Paris) between January 1,
2005, and January 1, 2011.
an external-validation cohort comprising patients who underwent
kidney transplantation at Foch Hospital (Suresnes, France) between
January 1, 2004, and January 31, 2010.
Clinical Data:
Anonymized data from national registries are prospectively entered
at specific time points for each patient (on day 0 and 6 months and 1
year after transplantation) and are updated annually thereafter.
Cases of acute clinical rejection, defined by deterioration in graft
function, proteinuria, or impaired function and histopathological
evidence of rejection, according to the consensus rules of the
international Banff classification criteria.
Histologic and Immunochemical Tests:
C4d staining was performed by means of immunochemical analysis
on paraffin sections with the use of polyclonal human anti-C4d
antibodies.
Detection and Characterization of Donor-Specific Antibodies:
All patients were tested for the presence of circulating DSA anti-HLA
antibodies in banked serum samples obtained at the time of
transplantation (day 0) and in serum samples obtained at the time of
the biopsy (1 year after transplantation or during an episode of acute
rejection in the first year after transplantation).
The presence of circulating donor-specific AntiHLA-A, -B, -Cw, -DR, –
DQ, and -DP antibodies was retrospectively determined with the use
of single antigen flow bead assays.
Patient with DSA anti-HLA antibodies were analyzed in a blinded
fashion at the University of Pittsburgh for the presence of C1q-binding
donor-specific anti-HLA antibodies with the use of single-antigen flow
bead assays.
Results:
Recipients In total, 1016 patients undergoing renal transplantation,
700 patients without circulating donor-specific anti-HLA antibodies,
239 patients with non–complement-binding donor-specific anti-HLA
antibodies, and 77 patients with complement-binding donor-specific
anti-HLA antibodies.
Kidney-Allograft Injury:
In the first year after transplantation, acute clinical rejection
developed in 171 patients: 96 patients had TCMR (56%) and 75 had
AMR (44%).
TCMR occurred in 14 of 77 patients with donor-specific anti-HLA
antibodies plus C1q-binding capacity (18%), in 30 of 239 patients with
donor-specific anti-HLA antibodies without C1q-binding capacity
(13%), and in 52 of 700 patients without donor-specific anti-HLA
antibodies (7%).
Patients with C1q-binding donor-specific anti-HLA antibodies had
more extensive microvascular inflammation and TG and higher
scores for graft PTC C4d deposition.
Patients with C1q-binding donor-specific antiHLA antibodies had a
lower estimated glomerular filtration rate (GFR) at 1 year.
Kidney-Allograft Survival:
Patients with donor specific anti-HLA antibodies had significantly
worse graft survival than patients without donor’s specific anti-HLA
antibodies (5-year graft survival after transplantation, 83% vs. 94%;
patients with C1qbinding donor-specific anti-HLA antibodies had the
poorest 5-year graft survival after transplantation (54%), as
compared with patients with non– C1q-binding donor-specific anti-
HLA antibodies and patients without donor-specific anti-HLA
antibodies (93% and 94%, respectively).
Determinants of Kidney-Allograft Loss:
Low estimated GFR at 1 year.
Interstitial fibrosis and tubular atrophy.
Glomerular and peritubular inflammation.
Transplant glomerulopathy.
Presence of complement-binding DSA.
Sensitivity Analysis:
By investigating associations separately in each study center and
according to kidney function and the timing of biopsies.
First, at both centers, patients with complement-binding donor-
specific anti-HLA antibodies had the lowest rate of graft survival.
Second, complement-binding donor-specific anti-HLA antibodies
after transplantation remained independently associated with graft
loss whether they were detected in specimens from protocol-specified
biopsies performed at 1 year or in specimens from biopsies
performed during acute rejection.
External Validation:
The external-validation cohort was composed of 643 patients.
Kaplan–Meier estimate of graft survival confirmed that patients with
complement-binding donor-specific anti-HLA antibodies had the
highest risk of graft loss as compared with patients with non–
complement binding donor-specific anti-HLA antibodies and patients
without donor-specific anti-HLA antibody.
Discussion:
In a cohort of 1016 carefully phenotyped kidney transplant recipients,
we observed that the presence of complement-binding donor-specific
antiHLA antibodies detected in the first year after transplantation was
an independent predictor of kidney-allograft loss more than 5 years
after transplantation and significantly improved individual risk
stratification for graft failure.
Type of study:
Prospective cohort study.
Level of evidence:
11
Excellent detailed analytical summary.
A prospective cohort with level of evidence :II
Allo -antibodies are still a major obstacle against successful transplantation. Anti-HLA antibodies, play significant role in kidney allografts failure due to allo immune response. Capillary C4d deposition has been considered as clue of antibody-mediated allograft damage and C1q properties of donor-specific anti-HLA antibodies may be specifically associated with AMR.
In this study,1016 patients from 2 centers with similar immunosuppressive protocols and induction regimens in both centers. They included CDC T and B cell negative cross match patients with follow up for 6 years. SAB Luminex was tested on day 0 and after 1 year with results of 700 patients had no DSA. 239 patients had non complement binding DSA and 77 patients with complement binding DSA. Kidney biopsies were done 1 year later or when clinical rejection was suspected. They concluded that complement binding anti HLA DSA were associated with lowest graft survival at the end of 6 years (54%) in comparison with 93% of those associated with non complement binding DSA.
Results support for the finding that C1q and C4d do not have similar predictive significance.C1q assay may aid identifying patients at risk, in spite of C4d negativity. Although C4d is specific ,it has insufficient sensitivity as an indicator of humoral rejection. The presence of complement-binding anti-HLA donor specific antibodies after transplantation is strongly associated with graft injury and loss. Considering this risk factor leads to better risk stratification for graft loss.
Very good highlighting the stronger risk significance of C1q HLA than C4d.
Please give a summary of this article
HLA antibody testing has undergone a sea-change in last 5 decades. From requirement of a negative crossmatch for transplant, we have reached a stage where transplants are being performed without a physical crossmatch. The journey of development of antibody testing is an interesting one.
The sensitivity of the original CDC crossmatch increased on using antihuman globulin. The sensitivity of antibody testing increased by use of flowcytometry and introduction of microparticle technology (FlowPRA and single antigen bead/ Luminex) further enhanced both sensitivity and specificity.
MFI values provided by the Luminex technology give a semiquantitative assessment of antibody levels, signifying a relative fluorescence of a single bead after binding to antibody. MFI values do not correlate with clinical outcomes.
The limitations of MFI include lack of clarity with respect to cut-off values; lack of standardization with respect to vendor, source of antigen and test reagents as well as interlaboratory differences. Various factors interfering with the assay include IgM HLA antibodies and complement split products giving rise to falsely low MFI, as well as denatured antigen giving rise to false positive results.
The power of pattern-recognition is important in antibody detection, represented by CREGs (cross-reactive groups) – multiple HLA antigens reacting with a single antibody, nowadays called epitopes, eplets etc. So, bead reactivity should be interpreted with
The avidity and degree of antibody binding to antigen depends on the conformation/ physical alteration of the antigen leading to variable MFI levels with same antibody, making it very difficult to interpret the results. hence eplets (discontinuous polymorphic regions) are important part of compatibility testing.
DSA detected by antigen binding in vitro might not bind to the antigen in vivo due to altered expression levels of the antigen, but might bind once the antigen expression increases as after an inflammatory stimulus. So, the MFI levels cannot be a marker of pathogenicity of the antibody.
SAB assays identifying complement binding antibodies did not conclusively prove their association with poor outcomes, especially in pre-transplant period. Post-transplant IgG3 antibodies have been shown to be associated with poor graft outcomes. IgG1 DSAs have been shown to have good graft outcomes. IgG may not fix complement in vivo due to presence of complement inhibitors like heparin sulphate and DAF, non-hexamerization of Fc fragment and insufficient IgG molecule binding.
Each case is different and center-specific multidisciplinary approaches for kidney transplantation is need of the day. HLA antibody detection and interpretation of its results needs to be done in light of the history and clinical parameters of the patient.
What is the level of evidence demonstrated by this study?
type 2 evidence study
What this type of this study?
Prospective cohort study
Fine but you missed the results and conclusion of value of C1q fixing antibodies in risk stratification
Please give a summary of this article
Despite the world wide advance in renal transplantations autoantibodies remain the major cause for graft loss. anti-HLA antibodies, plays a key role in the failure of kidney allografts due to alloimmune response, C4d deposition in renal capillaries has been considered the foot print of antibody-mediated allograft damage also complement fraction C1q properties of donor-specific anti-HLA antibodies may be specifically related to antibody mediated rejection.
Study summary
1016 patients from 2 centers were included in the study at 2005 2011 . Immunosuppressive protocols and induction regimens were same across both the centers. They included all CDC T and B cell negative cross match patients and followed them up for 6 years. The SAB Luminex was tested on day 0 and after 1 year. 700 patients had no DSA. 239 patients had non complement binding DSA and 77 patients with complement binding DSA. Kidney biopsies were done 1 year later or whenever the clinical nature of rejection was suspected. They concluded that complement binding anti HLA DSA were associated with lowest graft survival at the end of 6 years (54%) as compared to 93% of those associated with non complement binding DSA.
Coclusion
All results provide support for the finding that C1q and C4d do not provide equivalent predictive information. C1q testing may help identify patients at risk, despite C4d negativity, even C4d is specific,it lacks the sensitivity as an indicator of humoral rejection. The presence of complement-binding anti-HLA donor specific antibodies after transplantation is strongly associated with graft injury and loss and that incorporation of this risk factor improves risk stratification for graft loss.
Type of study : descriptive prospective cohort study
Level of evidence: II
Good
1.Summary of article- Presence of DSA is important and linked to inferior graft survival and antibody mediated rejection.But types of DSA ie whether it is complement binding or not, tell us better about graft survival and rejection.Presence of Complement binding DSA was associated with more acute rejection features like..Microvascular inflammation, Glomerulitis,Interstitial inflammation ,Presence of C4d in PTC as well as chronic rejection features like…Transplant glomeropathy,Interstitial fibrosis and tubular atrophy compared to DSA which are not complement binding. GFR at 1 yr was lowest in Complement binding DSA transplant compared to non complement binding DSA and No DSA transplant .Presence of Complement binding DSA drastically decrease graft survival compared to noncomplement binding DSA and No DSA transplant. Highlighting the fact that Presence of complement binding DSA may be more important than its quantity,,In this study Higher MFI was not associated with inferior graft loss compared to lesser values of MFI. 2. It was population based prospective study 3.Evidence level..2
Very good.
Type of this study is a prospective cohort study
Level of evidence is II
The article proposed the issue of assessment of the complement-binding capacity of DSA which can be used to reflect the power of DSA and the impact on graft survival.
Complement-fixing DSA can cause C4d positive ABMR, but it’s important to note that C4d deposition though specific but lacks sensitivity.
Complement fixing DSA are more clinically significant than non-complement fixing ones, note that the level of MFI may not correlate well to the significance of the antibodies. The above study elucidates that ABMR occurs more commonly in patients with C1q-binding DSA (those had significantly lower graft survival than those with non-C1q-binding DSA’s) when compared to non-C1q-binding DSA although in which HLA antibodies had inferior graft survival than those without DSAs.
Summary of article :
The Road to HLA Antibody Evaluation: Do Not Rely on MFI :
– Technological advances in HLA laboratory testing undoubtedly improved the sensitivity and specificity of HLA antibody assessment but not without introducing a set of challenges
regarding data interpretation. In particular, the introduction of solid-phase single-antigen bead (SAB) antibody assessment brought the belief that mean fluorescence intensity (MFI) was a quantifiable value.
A transplant candidate with a calculated panel-reactive antibody value of 100% who lives in Georgia was recently offered a virtual cross-match– negative, HLA-nonidentical kidney from a deceased donor in California. With no detectable donor-specific antibodies (DSAs) in a current serum sample (>>Under Construction: The Journey Begins :
Advances in techniques and technology paved the way to improved testing. Enhancement of the CDC assay with antihuman globulin made it possible to detect lower levels of antibodies than had been previously feasible. Next, the advent of flow cytometry led to the discovery and clinical implementation of the flow cytometric cross-match. Once again, sensitivity to detect even lower levels of antibodies was enhanced with the added benefit of simultaneous and independent assessment of both T cell and B cell crossmatches.
>>>Hard Hats Required: Controversies in HLA Antibody Assessment :
While multiplex Luminex technology (Luminex Corporation, Austin, TX) has provided a specific and sensitive platform to identify HLA antibodies, it is not flawless. A major point of contention revolves around results from SAB testing being reported as a numerical value referred to as mean fluorescence intensity (MFI).
>>>New Directions :
With the introduction of more sensitive and specific approaches to determine donor– recipient compatibility, the HLA community has reaffirmed the power of pattern recognition. In the mid 1960s–1970s, the term “cross-reactive groups” (CREGs) was used to describe the serologic phenomenon of multiple unique HLA antigens that reacted with a single antibody. Rodey and Fuller reported that it was more common for patients to produce a few antibodies to shared determinants among a group of antigens than to generate multiple individual specificities .
>>> Conformation of an Integral Component to Antibody Pathogenicity :
While there has been a strong interest in the quantification of HLA antibodies, there has been virtually no attention given to how or whether antigen conformation affects antibody affinity, avidity, and resulting MFI values. The following example illustrates that it is reasonable to consider that the degree and strength of antibody binding is influenced by antigen conformation.
>>> Antigens Versus Antibodies :
It is important to realize that detecting DSAs is predicated on a two-sided equation. On one side is the patient’s serum (antibody); on the other, the antigen it recognizes. When a DSA is identified, it is crucial to understand that microparticles have been coated with an amount of antigen designed to detect low levels of antibody, not to mimic the quantity of antigen on lymphocytes or tissues.
>>> Merge: “Functional” Methods to Assess Antibody Pathogenicity :
Several studies have focused on the complement fixing characteristics of HLA antibodies,
anticipating that those properties would be associated with their pathogenicity. SAB assays
were modified to assess which, if any, HLA antibodies in patient sera bound specific complement components (C1q, C3d, C4d) as a surrogate of complement activation in vivo. Published studies have been inconsistent, with some reporting strong associations between complement fixation and poor allograft outcome , while others found no correlations.
>>> Guides to Antibody Assessment :
Difficult and problematic cases cannot always be resolved with additional testing and/or sophisticated algorithms. Sometimes, there are no other tests to perform. For example, identical antibody presentations (e.g. antibodies to HLA-A*02 well below the established cutoff value) in three different individuals being considered for transplantation with an HLA-A*02–positive donor do not mean the same pathway will be taken for all three subjects.
Please give a summary of this article :
– Anti-HLA antibodies hamper successful transplantation, and activation of the com- plement cascade is involved in antibody-mediated rejection. We investigated whether the complement-binding capacity of anti-HLA antibodies plays a role in kidney-allograft failure.
– Assessment of the complement-binding capacity of donor-specific anti-HLA antibod- ies appears to be useful in identifying patients at high risk for kidney-allograft loss.
– In the United States and Europe, thousands of kidney transplants fail each year, and kidney-allograft failure is a major cause of end-stage renal disease, leading to in- creased morbidity, mortality, and costs.
– Since the pioneering discovery in 1969 that anti-HLA antibodies are lymphocytotoxic,activation of the complement cascade has been con- sidered to be a key component of antibody-medi- ated allograft rejection, and C4d deposition in renal capillaries has been considered the foot- print of antibody-mediated allograft damage.
– The capacity of anti-HLA antibodies to bind complement fraction C1q, which is the first step in activation of the classic complement cascade, determines the cytotoxic potential of these anti- bodies, and an assessment of their complement- binding capacity may be useful both for risk strati- fication and for diagnosis of antibody-mediated rejection.
– The median follow-up after transplantation was 4.8 years (range, 0.2 to 7.0). The median follow- up times were 3.9 years (range, 0.4 to 7.0) in pa- tients with C1q-binding donor-specific anti-HLA antibodies and 4.3 years (range, 0.2 to 7.0) in pa- tients with non–C1q-binding donor-specific anti- HLA antibodies.
– The inclusion of complement-binding donor-spe- cific anti-HLA antibodies in the reference model significantly improved its discrimination capacity.
-In conclusion, we systematically evaluated im- munologic characteristics before and after trans- plantation in a population-based sample of kid- ney-allograft recipients, incorporating the full spectrum of graft phenotypes. We found that the presence of complement-binding anti-HLA donor- specific antibodies after transplantation is strongly associated with graft injury and loss and that in- corporation of this risk factor improves risk stratification for graft loss.
References
1. Nankivell BJ, Kuypers DR. Diagnosis and prevention of chronic kidney al- lograft loss. Lancet 2011;378:1428-37.
2. Gaston RS, Cecka JM, Kasiske BL, et al. Evidence for antibody-mediated injury as a major determinant of late kidney al- lograft failure. Transplantation 2010;90: 68-74.
3. Sellarés J, de Freitas DG, Mengel M, et al. Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonad- herence. Am J Transplant 2012;12:388-99.
4. Organ Procurement and Transplanta- tion Network and Scientific Registry of Transplant Recipients 2010 data report. Am J Transplant 2012;12:Suppl 1:1-156.
5. Rapport 2011 de l’activité de prélève- ment et de greffe de l’Agence de la Biomé- decine (http://www.agence-biomedecine. fr/Rapport-annuel-2011).
6. Terasaki PI, Ozawa M. Predicting kid- ney graft failure by HLA antibodies: a pro- spective trial. Am J Transplant 2004;4:438- 43.
7. Hourmant M, Cesbron-Gautier A, Terasaki PI, et al. Frequency and clinical implications of development of donor-spe- cific and non-donor-specific HLA anti- bodies after kidney transplantation. J Am Soc Nephrol 2005;16:280412.
What this type of this study?
-Prospective cohort study .
What is the level of evidence demonstrated by this study?
Level II evidence.
1-Level of evidence:2
2-prospective cohort study.
3-Summary of complement-binding Anti -HLA Antibodies and kidney -Allograft Survival
Introduction:
Complement–binding anti-HLA Antibodies mediated rejection is associated with a high risk for kidney allograft loss., so the alloimmune response is a major determinant of late kidney allograft loss.
The capacity of anti-HLA Abs to bind complement fraction c1q is the first step in activities of the classical complement cascade, leading to ABMR so the c4d deposits in renal capillaries.
Methodology:
Clinical data;
Statistical analysis:
Results:
C1q binding DSAs were associated with aggressive microvascular inflammation. Transplant glomerulopathy, high score C4d deposition in the PTC.this aggressive pathology was not seen in those with non-c1q binding DSA, and those without DSA
Determinants of Kidney-Allograft Loss:
Discussion:
Limitation of the study:
Variability of MFI cut point between transplant centers, so it will not reflect the true result of this study
Conclusion:
Excellent
Summary of the article: The study highlights the importance of the Complement binding anti HLA DSA in causing graft injury and ABMR. C4d in the renal biopsy of the transplant kidney has been considered the foot print of ABMR due to their persistence of C4d stain in the renal biopsy. Assays are available to detect C1q which is the first step in the complement pathway activation leading on to C4d degradation product which is available as a special stain in the renal biopsy. 1016 patients from 2 centers at France were included in the study. Immunosuppressive protocols and induction regimens were same across both the centers. They included all CDC T and B cell negative cross match patients and followed them up for 6 years. The SAB Luminex was tested on day 0 and after 1 year. 700 patients had no DSA. 239 patients had non complement binding DSA and 77 patients with complement binding DSA. Kidney biopsies were done 1 year later or whenever the clinical nature of rejection was suspected. They concluded that complement binding anti HLA DSA were associated with lowest graft survival at the end of 6 years (54%) as compared to 93% of those associated with non complement binding DSA. They had concluded that this cohort study could pave way for more clinical trails on the usage of complement inhibitors like C1q esterase inhibitors and ecluzimab for renal transplant as lowering the complement levels means less C1q binding and less severity of ABMR.
One of the most important advances in trans- plantation medicine has been the recognition that anti-HLA antibodies are destructive.
Although anti-HLA antibodies are considered to be harmful, there is a wide spectrum of graft in- jury related to these antibodies, ranging from no recognizable damage to f lorid rejection.
activation of the complement cascade has been considered to be a key component of antibody-medi- ated allograft rejection, and C4d deposition in renal capillaries has been considered the foot- print of antibody-mediated allograft damage.
The capacity of anti-HLA antibodies to bind complement fraction C1q, which is the first step in activation of the classic complement cascade, determines the cytotoxic potential of these anti- bodies, and an assessment of their complement- binding capacity may be useful both for risk strati- fication and for diagnosis of antibody-mediated rejection.
We conducted a study to define the full spec-
trum of kidney-allograft injury according to the C1q-binding properties of donor-specific anti-HLA antibodies in a large population-based study and to determine whether assessment for the pres- ence of C1q-binding donor-specific anti-HLA anti- bodies after transplantation might improve risk stratification for kidney-allograft loss.
Results
Baseline Characteristics of the Kidney- Allograft Recipients
1016 patients undergoing renal transplantation were included in the main analysis. Three distinct populations were identified after transplantation, according to the presence or absence of donor-specific anti-HLA antibodies and complement-binding capacity: 700 patients without circulating donor-specific anti-HLA anti- bodies, 239 patients with non–complement-binding donor-specific anti-HLA antibodies, and 77 patients with complement-binding donor-specific anti-HLA antibodies.
Kidney-Allograft Injury
In the first year after transplantation, acute clin- ical rejection developed in 171 patients:
96 patients had T-cell–mediated rejection (56%) and 75 had antibody-mediated rejection (44%). T-cell– mediated rejection occurred in 14 of 77 patients with donor-specific anti-HLA antibodies plus C1q-binding capacity (18%),
in 30 of 239 patients with donor-specific anti-HLA antibodies without C1q-binding capacity (13%), and in 52 of 700 patients without donor-specific anti-HLA antibodies (7%) .Antibody-mediated rejection occurred in 37 patients with C1q-binding donor- specific anti-HLA antibodies (48%) and 38 patients with non–C1q-binding donor-specific anti-HLA antibodies (16%) .
Among the patients with C1q-binding donor- specific anti-HLA antibodies, 67 had microvas- cular inflammation (87%), 28 had tubular and interstitial inflammation scores of 2 or higher (on a scale of 0 to 3, with higher scores indicat- ing more severe abnormality) (36%), 18 had end- arteritis (23%), 17 had transplant glomerulopathy (22%), 30 had moderate-to-severe arteriosclerosis (39%), 23 had moderate-to-severe atrophy-scarring lesions (interstitial fibrosis and tubular atrophy) (30%), and 47 had C4d deposition in peritubular capillaries (61%). Patients with C1q-binding do- nor-specific anti-HLA antibodies had more ex- tensive microvascular inflammation and trans-
plant glomerulopathy and higher scores for graft peritubular capillary C4d deposition than both patients with non–C1q-binding donor-specific anti-HLA antibodies and patients without donor- specific anti-HLA antibodies .Stratified analyses revealed that these increases applied to samples from protocol-specified biopsies, per- formed at 1 year, and samples from biopsies performed during an acute-rejection episode in the first year .
Kidney-Allograft Survival
kidney-allograft survival according to donor-specific anti-HLA antibody status after transplantation. Patients with donor- specific anti-HLA antibodies had significantly worse graft survival than patients without donor- specific anti-HLA antibodies (5-year graft sur- vival after transplantation, 83% vs. 94%; P<0.001 by the log-rank test).
When patients with donor- specific anti-HLA antibodies after transplantation were subsequently categorized according to complement-binding capacity, patients with C1q- binding donor-specific anti-HLA antibodies had the poorest 5-year graft survival after transplantation (54%), as compared with patients with non– C1q-binding donor-specific anti-HLA antibodies and patients without donor-specific anti-HLA anti- bodies (93% and 94%, respectively. The risk of graft loss according to the donor-specific anti-HLA anti- bodies–C1q status at day 0 and the status after transplantation revealed that patients with C1q- binding donor-specific anti-HLA antibodies after transplantation had the highest risk of graft loss
Prediction of Kidney-Allograft Loss
the addition of complement-binding donor-specific anti-HLA antibodies to the reference model adequately re- classified patients at lower risk for graft loss and those at higher risk.
Discussion :
A sensitive detection method for C1q-binding anti-HLA antibodies yield findings in addition to those of the single-antigen flow bead test. Al- though the mean fluorescence intensity of donor- specific anti-HLA antibodies is widely used in clinical practice for risk stratification, this study
showed that C1q-binding donor-specific anti-HLA antibodies remained strongly associated with the risk of graft loss regardless of the mean fluorescence intensity of the antibodies.
C1q and C4d do not provide equivalent predictive information. C1q testing may help identify patients at risk, despite C4d .they found that C4d, although specific, lacks sensitivity as an indicator of humoral rejection. Assessment for complement-binding donor-specific anti-HLA antibodies may provide an early indication of the potential for complement-mediated injury, without the functional requirement for progression through the pathway to C4d deposition. Detection of complement-binding donor-specific anti-HLA antibodies may indicate which of the antibodies present have the capacity to activate the complement cascade,
potentially but not inevitably leading to C4d de- position and complement-mediated damage or antibody-mediated injury in vivo. Such findings do not rule out a possible role for complement- independent mechanisms in allograft injury me- diated by complement-binding donor-specific anti- HLA antibodies.
Type of study : descriptive prospective cohort study with analytic component
Level of evidence: II
The Road to HLA anti-body Evaluation: Do Not Rely on MFI
Introduction
In past CDCXM was the available tests before transplantation. It has less specificity( pts with +Ve test may have good outcome) and less sensitivity( pts with negative test may still have graft failure) .Therefore, more reliable test was needed to detect & identify clinically important HLA Abs
Under Construction : The Journey Begins
Hard Hats Required : Controversies in HLA Antibody Assessment
Be Prepared to Stop : Limitations
Re-routing : New Directions
‘Cross-reactive groups’ (CREGs)
Bumps in the road : Conformation – An integral component to antibody pathogenicity ?
Divided Highway : Antigens versus Antibodies
Merge: ‘Functional’ methods to assess antibody pathogenicity
Re-paving the Road : Guide to Antibody Assessment
Conclusion :
There is long way to go regarding HLA antibody detection and identification
New tests are needed but it is still not clear if these test will answer the old and future questions in this crucial matter in transplantation
1 – RESUME:
Complement-Binding Anti-HLA Antibodies and Kidney-Allograft Survival
Various studies over the past decade have indicated that the alloimmune response, mediated by anti-HLA antibodies, plays a key role in the failure of kidney allografts; this concept has been extended to heart, lung, and composite tissue transplants. Such a varied effect underscores the need to define distinct graft phenotypes and outcomes according to the presence or absence and characteristics of donor-specific anti-HLA antibodies after transplantation. Anti-HLA antibodies are lymphocytotoxic, activation of the complement cascade has been considered to be a key component of antibody-mediated allograft rejection, and C4d deposition in renal capillaries has been considered the footprint of antibody-mediated allograft damage. The capacity of anti-HLA antibodies to bind complement fraction C1q, which is the first step in activation of the classic complement cascade, determines the cytotoxic potential of these antibodies, and an assessment of their complementbinding capacity may be useful both for risk stratification and for diagnosis of antibody-mediated rejection.
This article is about one study in Paris at Necker Hospital and Saint-Louis Hospital between January 1, 2005, and January 1, 2011. The transplantation allocation system was identical for the three centers and followed the rules of the French national agency for organ procurement (Agence de la Biomédecine). All transplants were compatible with the ABO blood group. A negative result of cross-matching for IgG T-cell and B-cell complement-dependent cytotoxicity was required for all recipientes. It documented all cases of acute clinical rejection, defined by deterioration in graft function, proteinuria, or impaired function and histopathological evidence of rejection, according to the consensus rules of the international Banff classification criteria.
RESULTS:
Three distinct populations were identified after transplantation:
– patients without circulating donor-specific anti-HLA antibodies – 700
– patients with non–complement-binding donor-specific anti-HLA antibodies – 239
– patients with complement-binding donor-specific anti-HLA antibodies – 77
1 – Kidney-Allograft Injury
In the first year after transplantation, acute clinical rejection developed in 171 patients:
– 96 patients had T-cell–mediated rejection (56%)
– 75 had antibody-mediated rejection ( 44%)
– Patients with C1q-binding donor-specific anti-HLA antibodies had more extensive microvascular inflammation and transplant glomerulopathy and higher scores for graft peritubular capillary C4d deposition than both patients with non–C1q-binding donor-specific anti-HLA antibodies and patients without donor specific anti-HLA antibodies
– Patients with C1q-binding donor-specific antiHLA antibodies had a lower estimated glomerular filtration rate than did patients with non–C1q-binding donor-specific anti-HLA antibodies and patients without donor-specific anti-HLA antibodies
2 – Kidney-Allograft Survival
The median followup times were 3.9 years in patients with C1q-binding donor-specific anti-HLA antibodies and 4.3 years in patients with non–C1q-binding donor-specific antiHLA antibodies.
Patients with donor specific anti-HLA antibodies had significantly worse graft survival than patients without donor specific anti-HLA antibodies.
3 – Determinants of Kidney-Allograft Loss
The following independent predictors of graft loss were identified:
– low estimated GFR at 1 year:
– interstitial fibrosis and tubular atrophy
– glomerular and peritubular inflammation and transplant glomerulopathy
– presence of complement-binding donor specific anti-HLA antibodies after transplantation
DISCUSSION
Was observed that the presence of complement-binding donor-specific antiHLA antibodies detected in the first year after transplantation was an independent predictor of kidney-allograft loss more than 5 years after transplantation and significantly improved individual risk stratification for graft failure
2 – What is the level of evidence demonstrated by this study?
Evidence level 2
3 – What this type of this study?
This study is a prospective cohort
Development of Anti HLA antibodies leads to a poor graft outcome and whether complement binding adds to this deleterious affect has been investigated in this perspective cohort study of Alexander loupy and colleagues.
This was a multi center study conducted in Paris. A total of 1016 Patients were enrolled between January 1,2005 and January 1, 2011.
The participants were divided into two groups. As an external validation cohort, beneficiaries of organ transplants from a different transplant hospital were recruited. The anti-HLA antibodies and complement binding capabilities of all the patients were tested on days 0 and 1-year post-transplant, as well as during the moment of acute clinical rejection, in order to identify donor-specific antibodies. A biopsy of the kidney graft was performed one year after the transplant, or at the time of acute clinical rejection.
In the first year 171 patients developed acute clinical rejection, 96 patients TCMR and 75 ABMR.
TCMR patients were as follow: 14/77 with DSAs with C1q binding capacity, 30/239 with DSAs without C1q binding capacity, 52/700 without DSAs.
ABMR patients were as follow: 37/77patients with DSAs with C1q binding capacity, 38/239 patients with DSAs without C1q binding capacity.
Patients with DSAs with C1q binding capacity had increased risk of extensive microvascular inflammation and transplant glomerulopathy and higher scores for graft peritubular capillary C4d deposition and lower estimated GFR than both patients with non–C1q-binding donor-specific anti HLA antibodies and patients without donor specific anti-HLA antibodies.
C1q binding DSA (48%) was associated with severe ABMR than non-complement fixing antibodies(16%) and 5 yr Graft survival was less in patients with C1q binding DSA ( 54%) than non C1q binding DSA ( 93%) . Other factors that worsened graft outcome included increment in donor age, each one minute increase in cold ischemia time, deceased donor and retransplant Therapeutic agents targeting complement (e.g., a C5 inhibitor [eculizumab] or a C1 inhibitor) are being used in transplant recipients
Conclusion: the presence of DSA is associated with poor allograft outcome and also helps in risk stratification of patients for graft loss .
Introduction ;
Methodology ;
Study population ; After approval & obtained written concern
Clinical data ;
Histologic & immuno-chemical tests ;
Detection & Characterization of Donor-specific anti-bodies ;
Statistical analysis ;
Results ;
Determinant of kidney allograft loss ;
The independent predictors of graft loss were ;
Discussion ;
Limitations of the study ;
Conclusion ;
. The Road to HLA Antibody Evaluation: Do Not Rely on MFI
FlowPRA™ and SAB assays are used for the detection of HLA antibodies. FlowPRA™ is a screening test, one that qualitatively detects the presence/absence of HLA antibodies. SAB is a semiquantitative test used to determine antibody specificity.
-The results from SAB testing report as a numerical value referred to as mean fluorescence intensity (MFI). MFI values were never intended to quantify antibodies, nor was the Luminex-based test approved as a quantitative assay by the US Food and Drug Administration.
-MFI values reflect a given bead’s relative fluorescence without reference to a standard and fluorescence can be affected by many variables.
– The tendency is to correlate MFI values with clinical outcomes and to serially monitor their fluctuations as a measure of clinical status. Decreasing MFI values after a patient has been treated for antibody-mediated rejection (AMR) is interpreted as a proper clinical response, while no change (or an increase in value) is considered a therapy failure.
-If the MFI threshold is set too low, false positives may preclude a patient from receiving a compatible organ, while if the threshold is set too high, there may be an increased risk that a patient will experience early and/or irreversible AMR.
-There is no single value of MFI that corresponds to an unequivocal threshold above or below which organ transplantation is contraindicated or risk-free, respectively. As a result, cutoff MFI values can vary significantly by the organ transplanted and/or a transplant program’s practice.
-MFI variability is compounded by vendor, antigen source, test reagents, antibody-dependent complement activation, and intralaboratory variability.
– Mechanisms of assay interference include IgM HLA antibodies, wherein such antibodies compete with or block the binding of IgG HLA antibodies.
– False-positive reactivity can occur due to antibody binding to misfolded HLA proteins, commonly referred to as denatured antigens.
– Reactivity is attributed to the binding of antibodies to so-called cryptic epitopes, which under normal circumstances are considered to be antibody inaccessible. Such antibodies have a high prevalence among waitlist candidates (11–40%) likely explaining why some high-MFI DSAs were not associated with poor graft outcomes.
-Cross-reactive groups” (CREGs) were used to describe the serologic phenomenon of multiple unique HLA antigens that reacted with a single antibody.
– The degree and strength of antibody binding is influenced by antigen conformation.
– Microparticles have been coated with an amount of antigen designed to detect low levels of antibody.
-The most common de novo antibody produced posttransplantation is directed to HLA-DQ antigens (15) despite low expression of the DQ loci.
-The “pathogenicity” of an antibody cannot and should not be assumed based strictly on MFI levels.
– There are associations between IgG DSA subclass (in particular, IgG3) and poor clinical outcomes in the posttransplantation setting.
-A better understanding of the processes that drive alloantibody production in an individual is necessary to best characterize the pathogenicity of a given antibody.
The Road to HLA Antibody Evaluation: Do Not Rely on MFI
This article describes the techniques and technologies of HLA Abs testing starting from CDC enhancement with antihuman globulins to detect lower levels of Abs and the development of flow cytometry crossmatch techniques till the breakthrough of microparticles technologies introduction to practice.
Controversies in HLA Antibody Assessment
Although, multiple Luminex technology provided high specific and sensitive test for HLA Abs its report in numerical value as MFI become a mislead to clinicians’ interpretation of the results. The presentation of MFI as a numerical value gives the impression that this is a quantitative test – which is not-. Actually, MFI reflects the relative fluorescence of a given bead without a reference to a standard.
Limitations
MFI thresholds are variable between transplant centers and depending on a single test like MFI.if a given center chose a low threshold In order to decrease graft rejection the patient is prevented from receiving a compatible donor, in actual practice transplanted kidneys with very low threshold of MFI experienced poor graft outcome. On the other hand, transplanted patients with very high MFI threshold had excellent graft outcome!
So, why to sit these rigid cutoffs if their reflections on clinical outcome is not practical!
MFI variabilities can be caused by
1- Intra-laboratory variabilities caused by day-day variabilities including ambient temperature
2- Technologists dependent actions like pipetting, washing…etc.
3- Interference due to antibody-dependent complement activation in which the compliment split products bind to Ag-Ab complexes on the beads blocking secondary Abs binding resulting in diminished MFI values>> this may result on graft loss reported with low MFI
4- IgM HLA Abs competing against IgG HLA Abs binding sites cause the same results in point 3
5- Literature reported that sera from some patients contain Abs against both native and denatured HLA Ags (these denatured Ags result from manufacturing and coating of microparticles), normally Abs do not interact with these misfolded HLA but they are detected on the test resulting in high MFI>> this may explain why some patients with high MFI DSAs were not associated with poor graft outcome.
New Directions
CREG term introduced in 1960s to describe the phenomena of a single Ab reacts to multiple unique HLA Ags. Today, this observation was illustrated by Abs reaction to epitopes, eplets and conformational variances. We can consider Abs reacting to epitopes rather than HLA Ags this can lead to interpret non- DSAs as DSAs. (The target of DSAs is epitopes rather a whole HLA Ag)
Conformation—An Integral Component to Antibody Pathogenicity?
Actually, no attention is given on how Ag conformation affects Abs affinity, avidity and the resulting MFI values.
For example, Ab to Bw6 with high MFI except for B*46:01- and B*73:01-bearing beads which they have a single amino acid substitution in their epitops at position 75 (from glutamic acid to valine), interestingly HLA-C locus Ag had the same substitution (sharing Bw6/V75 epitop) resulting in low MFI also. This is an example of a single amino acid but this may be cause by several amino acids differences. Whatever the change, it collectively results in a physical alteration that affect MFI values.
Antigens Versus Antibodies
DSAs are detected by the interaction of the patient’s Abs with the Ags expressed in microparticles (which are designed to detect low titr Abs) but they are not expressive of the actual Ags load on lymphocytes or tissues. So, reacting Abs in vitro are not importantly representative of their interaction in vivo -although, inciting events like inflammation may renders the transplanted organ more susceptible to Abs binding. In conclusion, the pathogenicity of Abs cannot and should not be assessed solely on MFI values.
Functional” Methods to Assess Antibody Pathogenicity
Back to previous section, the pathogenicity of Abs may be assessed more practically by the Abs compliment fixing characteristics. So, SAB assays were modified to assess if Abs can bound compliment components (C1q, C3d, C4d) as a surrogate of complement activation in vivo. The results are debatable, were some proposed strong correlation and others no correlation at all. One large study, proposed that complement fixing DSAs were of clinical significance only on the posttransplant period.
Guides to Antibody Assessment
Each case should be assessed separately, Difficult cases cannot be assessed straightforward with algorithms or a single correct approach. The issue of HLA Abs detection, identification and interaction is still evoluting and may be or may be not one day evolute to the point where to reflect the patient’s true clinical interaction and outcome
Not all anti-HLA antibodies are associated with graft injury. This study hypothesized that their ability to bind complement plays a major role in their significance in causing graft injury. This study included 1016 patients and was conducted in two transplantation centers in Paris from 2005till 2011 . Screening of circulating DSA anti-HLA antibodies , their complement-binding capacity and graft injury phenotype were assessed. Complement binding DSA were significantly associated with less 5-year graft survival, high AMR , severe form of graft injury and more c4d deposition, compared to non–complement-binding DSA anti-HLA antibodies and patients without DSA. C1q-binding donor-specific anti-HLA antibodies remained strongly associated with the risk of graft loss regardless of the mean fluorescence intensity of the antibodies.This study postulated that the detection of complement-binding DSA anti- HLA antibodies in the first year post transplantation is an independent predictor of kidney-allograft loss more than 5 years after transplantation and significantly improved individual risk stratification for graft failure.
2b
cohort
The ROAD to HLAantibody evaluation: Do not rely on MFI
In this article, the advancement of technology and antibody detection was discussed. especially with the new solid-phase assay technology single antigen detection had very important information valuable in risk assessment. with this advancement in contrary to classic PRA evaluations even overseas or distant matching became available. although the mean fluorescence intensity (MFI) used here was not intended to be a quantitative measurement in practice it was sealed as such. there is variability from center to center and the other is a handicap. some antibodies with low intensity may disappear as they bind to multiple beads to become undetectable or detected but are considered as non-significant as they are below the threshold.
The problem with MFI is that if the threshold is set to a very low level this may preclude compatible patients from receiving organs. on the contrary setting, it is too high will lead to increased rejection.
the phenomenon of cross-reactive groups described in the 1960s-1970s now describes as eplets, epitopes is another challenge. public epitopes shared between multiple HLA antigens will lead to rejection although not presented in the report or may be presented with low intensity…
another poşnt is the relation between AB-AG; in the test, the beads are coated to detect very low levels of antibodies which may not be the parallel case in vivo and vice versa.
The type of IgG is also important. In vitro IgG1 and IgG3 fix complement to a higher degree (Ig2 less, IgG4 may not). In vive the same extent of effect may not bee seen. Hexamerisation of IgG Fc fragment and engagement of IgG antibodies may not correlate with their concentration. presence of IgG antibodies may not be sufficient to sustain or to start the complement activation cascade.
same antibody even with similar concentrations but in different recepients is not expected to have same effect.
An overview of this article.
Graft damage is related to the presence of anti-HLA antibodies. It has been shown that they bind to the C1q component of complement. In order to determine the function of complement binding capability of anti-HLA antibodies in renal graft prognosis, an open-label, prospective cohort research was designed and implemented.
A total of 1016 transplant patients from two transplant facilities in Paris were involved in the research between January 2005 and January 2011. The participants were divided into two groups. As an external validation cohort, beneficiaries of organ transplants from a different transplant hospital were recruited. The anti-HLA antibodies and complement binding capabilities of all the patients were tested on days 0 and 1-year post-transplant, as well as during the moment of acute clinical rejection, in order to identify donor-specific antibodies. A biopsy of the kidney graft was performed one year after the transplant, or at the time of acute clinical rejection.
The existence of DSAs that bind to C1q in the sera of patients with DSA was investigated.
Patients were selected based on the presence or absence of DSA as well as their complement binding capability.
Detection of complement binding DSA in the first year after transplantation was an independent predictor of graft loss 5 years after transplantation and considerably improved risk stratification for graft failure, according to the study.
Those that suffered from complement binding DSA had graft damage, which was characterized by microvascular inflammation and C4d deposition.
The presence of C1q binding DSA was shown to be significantly linked with the likelihood of graft loss, independent of the MFI of the antibodies.
The detection of complement binding DSA may suggest the possibility of complement-mediated damage before development along the route leading to C4d deposition in the body.
The capacity to bind complement may be essential since novel therapeutic drugs such as the C5 inhibitor (eculizumab) and the C1 inhibitor (adalimumab) that target complement are increasingly being utilized in kidney transplant patients.
The type of the study: a prospective cohort study with a defined sample size
the level of evidence: Evidence at the second level
summary of additional article
MFI mean fluorescence intensity has some limitations make it not a good indicator for transplantability;
It has no universal cut off value , above which there is a contraindication for transplantation, as it is dynamic vary from day to day and from center to center, it is of value in showing response to treatment of rejection.
There are a cases transplanted against high MFI showed favorable outcome with no episode of rejection, and other cases are transplanted with low MFI showed occurrence of episodes of rejection with non-favorable graft outcome.
Interlaboratory factors can lead to variable MFI accounts for more than 50% of its variability, it is affected by reagent used, kits itself, daily variation of MFI and also technologist worked (operator dependent), all interlaboratory factors can lead to false positive of false high MFI which negatively impacts the decision of transplantation.
We should be epitope minded
CREGs cross reactive groups described reactivity of multiple HLA antigens with single antibody.
Rejected transplanted graft happens even with matched HLA antigen , this will be clarified by shared antigen in single antibody , so the match should be directed to the level above the level of antigen like epitope (specific epitope ) which will improve graft survival and outcome.
The road to HLA antibody evaluation: Do not rely on MFI
Introduction:
HLA antibody testing has undergone a sea-change in last 5 decades. From requirement of a negative crossmatch for transplant, we have reached a stage where transplants are being performed without a physical crossmatch. The journey of development of antibody testing is an interesting one.
Under construction: The journey begins.
The sensitivity of the original CDC crossmatch increased on using antihuman globulin. The sensitivity of antibody testing increased by use of flowcytometry and introduction of microparticle technology (FlowPRA and single antigen bead/ Luminex) further enhanced both sensitivity and specificity.
Hard hats required: controversies in HLA antibody assessment
MFI values provided by the Luminex technology give a semiquantitative assessment of antibody levels, signifying a relative fluorescence of a single bead after binding to antibody. MFI values do not correlate with clinical outcomes.
Be prepared to stop: limitations
The limitations of MFI include lack of clarity with respect to cut-off values; lack of standardization with respect to vendor, source of antigen and test reagents as well as intralaboratory differences. Various factors interfering with the assay include IgM HLA antibodies and complement split products giving rise to falsely low MFI, as well as denatured antigen giving rise to false positive results.
Rerouting: New directions
The power of pattern-recognition is important in antibody detection, represented by CREGs (cross-reactive groups) – multiple HLA antigens reacting with a single antibody, nowadays called epitopes, eplets etc. So, bead reactivity should be interpreted with respect to epitopes and not antigens.
Bumps in the road: Conformation – an integral component to antibody pathogenicity?
The avidity and degree of antibody binding to antigen depends on the conformation/ physical alteration of the antigen leading to variable MFI levels with same antibody, making it very difficult to interpret the results. hence eplets (discontinuous polymorphic regions) are important part of compatibility testing.
Divided highways: antigens versus antibodies
DSA detected by antigen binding in vitro might not bind to the antigen in vivo due to altered expression levels of the antigen, but might bind once the antigen expression increases as after an inflammatory stimulus. So, the MFI levels cannot be a marker of pathogenicity of the antibody.
Merge: “functional” methods to assess antibody pathogenicity
SAB assays identifying complement binding antibodies did not conclusively prove their association with poor outcomes, especially in pre-transplant period. Post-transplant IgG3 antibodies have been shown to be associated with poor graft outcomes. IgG1 DSAs have been shown to have good graft outcomes. IgG may not fix complement in vivo due to presence of complement inhibitors like heparin sulphate and DAF, non-hexamerization of Fc fragment and insufficient IgG molecule binding.
Repaving the road: guide to antibody assessment
Each case is different and center-specific multidisciplinary approaches for kidney transplantation is need of the day. HLA antibody detection and interpretation of its results needs to be done in light of the history and clinical parameters of the patient.
· summary of additional article about MFI
Although Luminex SAB is the standard used in HLA typing, many limitations exist.
· SAB and MFI were never designed to quantify DSA. However, their trend can be followed to determine response to desensitization protocol or anti-rejection therapy in AMR.
· Q. why not to use MFI as indicator for translantability (CI to transplant or predictive for the prognosis)?
o No defined cut off level for +ve or -ve tests (not standardized between different centers).
o Low threshold may hinder transplantability.
o Higher threshold may carry risk of early AMR.
o No direct relation was found between MFI and graft outcome. However, cases with low MFI had poor prognosis and others with higher MFI survived well.
o Marked intra-laboratory differences depending up on:
§ Temp and PH of reaction.
§ Methodology and amount of fluroscenated antibodies added.
§ In case of complement fixation antibody reaction (complement binding may decrease or interfere with anti HLA IgG antibodies (false -ve results).
§ Presence of IgM antibodies and anti-epitopes antibodies can give positive results invitro. However, they don’t usually reflected on in-vivo reaction and graft outcome.
§ Recent trend toward anti-epitope antibodies.
§ Reaction to bead coating with antigen differ from in-vivo reaction with lymphocytes antigens, so +ve DSA in-vitro may fail to react with cells in-vivo.
§ Although complement fixing antibodies ( IgG1, 3 ) were thought to have worse prognosis and early occurrence in early post transplant period than non complement fixing (IgG 2, 4 antibodies), some reports show conflicting results. This can be explained by the difference in reaction between invitro testing and actual invivo immune reaction.
· So , no single best test and no single approach in antibody testing..
· Individualized approach and case by case management is the ideal approach.
· Never to depend on snapshot to determine recipient risk, instead, tailor treatment to history, previous transplant and prior sensitization.
Complement-Binding Anti-HLA Antibodies and Kidney-Allograft Survival
Introduction
Alloimmune response is a major cause of renal transplant failure. Alloimmune response mediated by anti-HLA Abs play a key role in failure of kidney graft and these Abs response varies between no effect at all to destructive outcomes on the allograft.
Anti HLA Abs are lymphocytotoxic and cause the activation of compliment cascade by binding to C1q – which is the first step in activating the classic pathway- leading to ABMR, this is why C4d deposits in renal capillaries is a landmark of ABMR.
This study purpose was to asses if C1q binding properties of DSAa may improve risk stratification for kidney-allograft loss.
Methodology
The data collected prospectively from three different centers with all patients are ABO compatible and with negative cross match for IgG T cell and B cell complement dependent cytotoxicity and patients were followed at 66 months, 1 year and every year after for a total of five years.
Acute clinical rejection was defined by deterioration in graft function, proteinuria, or impaired function and histopathological evidence of rejection, according to the consensus rules of the international Banff classification criteria. Immunosuppression protocols and treatment of allograft rejection episodes after transplantation were similar among the centers.
854 biopsies were taken from patients with no evidence of acute rejection at 1 year and 171 biopsies were taken within 1 year from patients with evidence of acute rejection. All biopsies were categorized by Banff score by three different pathologists who were blinded to the status of patients’ HLA As, C1q bonding status and clinical score. Also, C3d staining was performed by immunochemical analysis.
Detection and Characterization of Donor-Specific Antibodies
patients were tested for the presence of DSAs at day 0 and at time of biopsy (either at 1 year or at time of rejection in the first year) using single Ag flow bead assays on Luminex platform. Patients with identifiable DSAs were analyzed for the presence of C1q-binding donor-specific anti-HLA antibodies with the use of single-antigen flow bead assays
Results
Baseline Characteristics of the Kidney-Allograft Recipients
A total of 1016 patients were included at the time of transplantation in three groups
1- Without circulating DSAs n=700
2- Non complement binding DSAs 239
3- Complement binding DSAs 77
Kidney-Allograft Injury
In the first year 171 patients developed acute clinical rejection, 96 patients TCMR and 75 ABMR.
TCMR patients were as follow: 14/77 with DSAs with C1q binding capacity, 30/239 with DSAs without C1q binding capacity, 52/700 without DSAs.
ABMR patients were as follow: 37/77patients with DSAs with C1q binding capacity, 38/239 patients with DSAs without C1q binding capacity.
Patients with DSAs with C1q binding capacity had increased risk of extensive microvascular inflammation and transplant glomerulopathy and higher scores for graft peritubular capillary C4d deposition and lower estimated GFR than both patients with non–C1q-binding donor-specific anti HLA antibodies and patients without donor specific anti-HLA antibodies.
Kidney-Allograft Survival
Patients with DSAs with C1q binding capacity had the poorest rates of 5-year graft survival followed by DSAs without C1q binding capacity and patients without DSAs.
Determinants of Kidney-Allograft Loss
Independent predictors of graft loss yielded from analysis include: low estimated GFR at 1-year, interstitial fibrosis and tubular atrophy, glomerular and peritubular inflammation and transplant glomerulopathy, and the presence of complement-binding donor specific anti-HLA antibodies after transplantation (theses DSAs were associated with the same graft survival regardless of their MFI)
Sensitivity Analysis
At both centers, patients with complement binding DSAs had the lowest rate of graft survival, complement-binding donor-specific anti-HLA antibodies after transplantation remained independently associated with graft loss whether they were detected in specimens from protocol-specified biopsies performed at 1 year or in specimens from biopsies performed during acute rejection in the first year, C1q-binding DSAs C1q-binding
Discussion
The presence of complement binding DSAs in the first year after transplant were independently associated with allograft loss at 5 years follow up and significantly improved individual risk stratification for graft failure.
This graft injury phenotype was characterized by microvascular inflammation and complement split-product C4d deposition stratifying patients according to their complement binding capacity -not only to the presence or absence of DSAs- identified an additional group of patients with an increased risk of graft loss.
C1q-binding donor-specific anti-HLA antibodies remained strongly associated with the risk of graft loss regardless of the mean fluorescence intensity of the antibodies.
The presence of C1q and C4d do not provide the same prognostic utility. C1q testing may help identify patients at risk, despite C4d negativity.
Assessment for complement-binding donor-specific anti-HLA antibodies may provide an early indication of the potential for complement-mediated injury, without the functional requirement for progression through the pathway to C4d deposition
HLA antibody evaluation
CDCXM with antihuman globulin allowed detection of low level of antibodies.
FCXM provide simultaneous and independent assessment of both T cell and B cell crossmatch.
Flow PRA: A screening test, qualitatively detects the presence and/or absence of HLA antibodies.
SAB assay: Semi-quantitative and detect and identify antibodies with higher specificity and sensitivity.
Controversies in HLA antibody assessment:
Although MFI is reported as numerical value, It wasn’t intended to quantify antibodies and SAB assay is not approved as quantitative test.
MFI reflects fluorescence without reference to standard.
Limitations:
There is no cutoff value at which organ transplantation is contraindicated or below it the transplant is considered risk free.
Intra-lab. variability of MFI results.
Reported MFI values are influenced by technologist dependent actions
MFI values may be falsely low due to interfering factors as complement split products and IgM HLA antibodies.
False positive reactivity may occur due to denatured antigens.
New directions is to consider antibody reactivity to be epitope directed and not t individual HLA antigen
Antibody pathogenicity:
The degree and strength of antibody binding may be influenced by antigen conformation which may be affected by differences in protein sequences.
The pathogenicity of antibodies can’t be determined according to MFI level.
Functional methods to assess antibody pathogenicity:
Complement fixing ability of antibodies may be used to assess their pathogenicity, but results of studies were controversial regarding the association between complement fixing ability and poor graft outcome, some studies suggested that antigens can fix complement in vitro but may not do so in vivo and complement independent antibody mediated rejection may occur.
Antibody assessment should be individualized according to each recipient.
In this article the author have investigated the role of complement binding capacity of anti HLA antibodies in allograft rejection. Graft injury due to Anti HLA antibodies can be variable ranging from no damage to significant rejection. Complement fixing DSA which can be detected by C1q assay ,can cause both C4d positive ABMR and C4d negative ABMR .It is more important than non-complement fixing DSA, however, it is not correlated to intensity of DSA.
Current study enrolled 1016 patients from two transplantation centers in Paris between January 1, 2005, and January 1, 2011 , speculated C1q binding DSA (48%) was associated with severe ABMR than non-complement fixing antibodies(16%) and Graft survival was less in patients with C1q binding DSA ( 54%) than non C1q binding DSA ( 93%) . Other factors that worsened graft outcome included increment in donor age, each one minute increase in cold ischemia time, deceased donor and retransplant Therapeutic agents targeting complement (e.g., a C5 inhibitor [eculizumab] or a C1 inhibitor) are being used in transplant recipients
In conclusion, the presence of DSA is associated with poor allograft outcome and also helps in risk stratification of patients for graft loss .
Type of study: descriptive prospective cohort study with analytic component
Level of evidence: II
This article focus on role of complement binding Anti HLA Ab detection on graft survival.
It’s prospective cohort study done by Alexandre et al, in two transplant centers in Paris from first of January 2005 to first January 2011 with fallow up transplanted patients up to April 2012; all selected patients are ABO compatible & CDC T / B cells are negative. it’s a comparison study between 3 groups, patients had complement binding DSA anti HLA Ab and patients with non complement DSA anti HLA Ab and patients with out DSA HLA Ab. Those patients are screening clinical/ Functional/ histologically and immunological. They found that patients with complement binding DSA anti HLA b had quadruple risk of graft loss & AMR then patients with non complement binding DSA anti HLA Ab and non DSA Ab regardless MFI. they show evidence of micro vascular inflammation and deposits of Cd4 complement with graft capillaries. Anti HLA Ab are lymphotoxic and lead to activation of complement by binding of anti HLA to C1q. that’s why it’s important to screen for presence of C1q binding to DSA HLA Ab to improve outcome of graft. In this study clinical data collected prospectively at day0 / 6 month/ 1 year post transplant and then annually, also documented patients with acute rejection who developed deteriorated renal function/ proteinuria/ patients with histological evidence according to banff classification.
detection and characterization of DSA Ab: all patients tested for presence of circulating DSA in serum at Day 0 and at day of biopsy after one year of transplant or during attacks of acute rejection. circulating DSA for HLA A B Cw DR DQ Dp determined with use of single antigen flow bead on luminex platform. serum of C1q binding DSA anti HLA Ab with use of single antigen flow bead obtained.
Results of 3 groups of patients assessed according to presence or absence of DSA anti HLA Ab from total 1016 patients showed 700 with out circulating DSA/ 239 with non binding complement DSA anti HlA antibodies and 77 with complement binding DSA anti HLA antibodies. they show T cell mediated rejection & AMR and microvascular inflammation more in patients with DSA and C1q binding rather than patients with non C1q binding DSA anti HLA Ab. Also histological changes with more extensive micro vascular inflammation and transplant glomerulopathy and high score of peritubular capillary C4d deposits more in patients with C1q binding and DSA HLA Ab rather than other s; Also noticed decrease GFR level in those patients.
Kidney allograft survival rate are lower around 3.9 years in patients with C1q binding DSA in comparison with non specific C1q binding anti HLA Ab where those average survival rate 4.8 years. So patients with C1q binding DSA anti HLA antibodies are at risk of allograft rejection regardless of MFI high or low.
Eculizumab are C5 inhibitors or C1 inhibitors promise to use in kidney transplant to reduce risk of allograft rejection.
Q2: level 2
Q3: This study is prospective cohort study
I. Complement-Binding Anti-HLA Antibodies and Kidney-Allograft Survival
Anti-HLA antibodies are associated with graft injury. They have been shown to bind to C1q fraction of complement. A prospective cohort study was planned with the aim to identify the role of complement binding capacity of anti-HLA antibodies in renal graft prognosis.
The study group included a cohort of 1016 transplant recipients from 2 transplant centers in Paris between January 2005 and January 2011. Transplant recipients at another transplant center were taken as external validation cohort. All the patients were screened for donor-specific anti-HLA antibodies and their complement binding capacities at day 0 and 1 year post-transplant or at the time of acute clinical rejection. Kidney graft biopsy was done at 1 year post-transplant, or at the time of acute clinical rejection.
Out of 1016, 239 patients had non C1q binding DSA and 77 had C1q binding DSA. T cell mediated rejection was highest in C1q DSA group. C1q DSA group was associated with 3 times higher AMR than non C1q binding DSA group and had least GFR at 1 year among the 3 groups. The 5 year graft survival was least with the C1q DSA group (54% versus 93%^for non complement binding DSA and 94% for DSA negative patients).
The C1q DSA group was associated with higher and more severe rejection with microvascular inflammation, increased C4d positivity and increased graft loss. C1q DSA had strong association with graft loss without any relation to their MFI. So, presence of complement binding DSA can be taken as an early predictor for complement mediated injury progressing to AMR. Hence, these antibodies should be considered as a risk factor for AMR while assessing the prognosis of graft kidney.
Level II evidence
Prospective cohort study
# Please give a summary of this article
* This study to investigate whether the complement binding capacity of anti-HLA antibodies plays a role in kidney allograft failure.
* In this study they enrolled patients who received kidney allografts at two transplantation centers in a population-based study.
* Patients were screened for the presence of circulating DSA and their complement binding capacity, Graft injury phenotype and the time to kidney allograft loss were assessed.
* The results is that
– The primary analysis included 1016 patients.
– Three distinct populations were identified after transplantation, according to the presence or absence of DSA
and complement-binding capacity:
– 700 patients without circulating DSA.
– 239 patients with non complement bindin DSA.
– 77 patients with complement-binding DSA.
# In the first year after transplantation, acute clinical rejection developed in 171 patients.
# 96 patients had T-cell mediated rejection (56%).
# 75 had antibody mediated rejection (44%).
* T-cell mediated rejection occurred in 14 of 77 patients with DSA plus C1q binding capacity (18%).
* In 30 of 239 patients with DSA but with out C1q binding capacity (13%).
* In 52 of 700 patients with out DSA(7%).
# Antibody-mediated rejection :
*Occurred in 37 patients with C1q binding DSA(48%).
* 38 patients with non C1q binding DSA(16).
# Among the patients with C1q-binding DSA :
* 67 had microvascular inflammation (87%).
* 28 had tubular and interstitial inflammation more severe abnormality (36%).
# Patients with C1q-binding DSA had more extensive microvascular inflammation , transplant glomerulopathy and high C4d deposition than both patients with non C1q binding DSA and patients with out DSA .
# Patients with C1q-DSA had a lower estimated glomerular filtration rate (GFR) at 1 year than patients with non C1q binding DSA and patients without DSA.
** After transplantation ( 5-year graft survival) :
# patients with DSA had significantly worse graft survival than patients without DSA , 83% vs. 94%; (P<0.001)
#. Patients with DSA were subsequently categorized according to complement binding capacity:
– Patients with C1q binding DSA had the poorest 5 year graft survival (54%), as compared with patients with nonC1q binding DSA.
# The risk of graft loss according DSA C1q status at day 0 and the status after transplantation revealed that they had the highest risk of graft loss .
# patients with C1q binding DSA had similar graft survival, regardless of whether the mean fluorescence intensity was
low or high.
# Conclusion:
– presence ofcomplement binding anti HLA donor specific antibodies after transplantation is strongly associated with graft injury and loss and that in corporation of this risk factor improves risk strati-
fication for graft loss
-The study gives a way for future therapeutic implications, agents targeting complement are increasingly being used in practice.
# What is the level of evidence demonstrated by this study?
LEVEL 2 evidence study.
# What this type of this study?
Prospective cohort study.
The road to HLA antibody evaluation:
The journey begins
Advances in techniques and technology paved the way to improved testing. These include adding antihuman globulin to DCD testing then the use of FCXM.
The game-changing moment in detecting HLA antibodies was the introduction of microparticle technology, including flow PRA & SAB assays. They are highly sensitive and specific, but these features have their challenges.
Controversies in HLA antibody assessment:
The primary concern about the result of SAB testing is that being a numerical value referred to as MFI. MFI reflects a given bead’s relative fluorescences without reference to a standard; many variables can affect this fluorescence.
Limitations:
New directions:
From the mid-1960 to 1970, the term cross-reactive groups were used to describe the phenomenon of multiple unique HLA antigens that reacted with a single antibody.
Today, the terminology changed to epitopes, epletes. With this change, what was previously known as third-party antibodies are now considered DSA.
Conformation- an integral component to antibody pathogenicity
The degree of antibody binding is influenced by antigen conformation, affecting the MFI value.
Antigens versus antibodies:
DSA that binds a bead in vitro may fail to attach to the corresponding antigen in vivo because its expression is too low. In vivo, a lower level of HLA antigen expression compared with beads could provide a target to which the DSA can bind. Therefore, the pathogenicity of an antibody cannot and should not be assumed strictly on MFI levels.
Functional methods to assess antibody pathogenicity:
Most of the studies focused on two things: complement-fixing and IgG subclass.
The largest study evaluating complement-fixing DSA with poor graft survival found a clinical significance only post-transplantation.
Studies found that not all sera containing potent complement-fixing antibodies can fix complement in vitro; the explanations for this:
Guides to antibody assessment
Challenging cases need a multidisciplinary team to outline the immunological complexities of particular donor-recipient pair and to discuss issues that typically one or more of the participants do not consider.
So the road toward HLA antibody detection and identification is still under construction.
The Road to HLA Antibody Evaluation: Do Not Rely on MFI
HLA antibodies detection Journey:
– Starting by CDC XM and because of lower sensitivity and specificity, human anti- goblin was added to increase the sensitivity (Anti-Human Globulin (AHG) augmented CDC), followed by FCM XM which provide good sensitivity an specificity when compared with CDC and AHG- augmented CDC and lastly DSA detection using luminex technique which is highly sensitive and specific technique for detection of HLA antibodies either donor on non-donor specific
Significance of DSA MFI level
– Luminex gives a quantitative assessment of the strength of DSA through measurement of MFI, the significance of MFI and its correlation to graft outcome is debatable which make the setting of certain threshold for significant MFI is extremely difficult, this may be explained by the following facts :
1- Different thresholds for detection of DSAs were set with variable MFI cutoffs so false negative and positive results can occur due to this variation, this means a positive result in one center is negative in another (interlaboratory variability).
2- Intra – laboratory variability due to reagents also affects the result of DSA MFI
3- The DSA MFI doesn’t correlate well with XM, It may predict negative XM (if it is below threshold) but not positive ones (have negative predictive value). Thus some patients may have high MFI and negative XM
4- Significance may differ according to HLA antigen to which antibody is directed, so significant MFI for DSA directed to HLA class I may differ from HLA directed to class II
5- The MFI doesn’t correlate well with graft outcome, some patients may have DSA with high MFI (> 10000) and have acceptable graft survival, others may have very low DSA (MFI 100) and develop graft loss
6- DSA response to treatment indicated by decreasing MFI may be associated with better graft survival
7- Complement fixing AB (determined by the C1q assay) are proved by some studies but not all to be more clinically significant than non-complement fixing AB , other studies reported this significance in denovo DSA and not performed DS, also complement-fixing ability is not correlated well with the intensity of DSA, this means that there may be a DSA with high intensity, but is not complement-fixing.
8- Expression of HLA antigen on kidney tissue may affect the significance of DSA MFI, for example if DSA directed to antigen with a low expression on kidney tissue may result in high MFI invitro, while invivo it may be either nonsignificant in some cases but in others become significant due to increased expression of this antigen invivo.
9- False positive result can occur due to :
• Denatured antigens attached to beads, leading to alteration in protein configuration
• Sharing of public epitopes among several antigens (HLA molecule-based matching). Epitope matching which includes the use of epitopes as the focus in HLA typing (epitope-based matching) eliminate this false-positive results this is called the highly specific SAB system
10- False-negative results can occur due to :
• High levels of IgM that can bind to antigen beads and prevent binding to actual IgG-DSA, this is called prozone efect or hook effect
• Very high level of DSA that agglutinate so fail to bind to the beed,
• Appearance of antigenic epitope on multiple beads, thus DSA bind to multiple beads thus diluted giving false-negative result, this will be detected by SAB.
Complement-binding anti-HLA antibodies and kidney –allograft survival
Introduction
It is well established that anti-HLA antibody production leads to kidney allograft failure.
The clinical outcomes of these antibodies range from causing no damage to severe allograft injury.
This varied difference in injury caused by these antibodies led the authors to postulate that this could be related to the difference in complement binding capacity of these antibodies.
C4d deposition in glomerular capillaries is considered to be the biological footprint of ABMR in the allograft.
Binding of antibodies to C1q is the first and perhaps most important step in the activation of the classical complement pathway.
Objectives
To define the complement binding capacity of anti-HLA antibodies.
To define patterns of allograft injury according to the C1q binding capacity of ant-HLA DSA
To determine utility of C1q binding DSA in risk stratification of kidney-allograft loss.
Methods
Prospective cohort study from January 2005 to January 2011
Conducted in France at two hospitals (Necker and Saint-Louis Hospitals)
Study population: All ABO compatible, and CDC T and B negative patients who underwent kidney transplantation during the study period
Clinical data was collected from the national registries.
Acute clinical rejection was defined by; Deterioration in graft function, proteinuria, and histological evidence of rejection according to Banff criteria.
Patients were treated with similar immunosuppression and rejection protocols
Biopsy results from 1 year protocol biopsies and for cause biopsies were used. C4d staining was done on all biopsies.
All patients were tested for anti-HLA DSA on pre-transplant stored blood samples and at the time of biopsy.
Results
1016 patients were included in the analysis.
700/1016 no DSA
239/1016 non-complement binding DSA
77/1016 complement (c1q) binding DSA
Kidney allograft injury
171/1016 had acute clinical rejection
96/171 (56%) had TCMR, 75/171 had ABMR
14/77 had TCMR + C1q binding DSA
30/239 had TCMR + non-C1q binding DSA
52/700 had TCMR + no DSA
37/77 had ABMR + C1q binding DSA
38/239 had ABMR + non-C1q binding DSA
Patients with C1q binding DSA were more likely to have;
✓ Extensive Microvascular inflammation
✓ Severe score for tubular and interstitial inflammation
✓ Interstitial fibrosis and tubular atrophy
✓ Severe transplant glomerulopathy
Allograft survival
Patients with DSA had worse graft survival than those without.
Patients with C1q binding DSA had the worst allograft survival.
Predictors of graft loss;
1. Low 1 year GFR
2. Interstitial fibrosis and tubular atrophy
3. Transplant glomerulopathy
4. C1q binding DSA
Conclusion
This has study has convincingly demonstrated the deleterious effects of DSA in terms of causing worse graft inflammation and loss of function, but most importantly the worst effects of DSA are seen with the C1q binding DSA.
The Road to HLA Antibody Evaluation: Do Not Rely on MFI.
Introduction:
Positive CDC cross match was a contraindication for Transplantation but some patient got good graft survival and vice versa some patients with negative CDC cross match had many attacks of rejections with poor graft outcome so we are looking for another reliable method to detect clinical relevant of HLA antibody.
The Journey Begins:
To improve CDC sensitivity ,AHG added to detect lower level of HLA antibody then flow-cytometry technique started with high sensitivity and with independent assessment of both T &B Cell cross match. Now SAB assay provided with high sensitivity and specificity in detection and identification of HLA class I&II.
Controversies in HLA Antibody Assessment:
MFI is a numerical value of SAB result which reflects a given bead’s relative fluorescence without reference to a standard.
Which used to assess antibody strength and to correlate number with clinical outcomes especially after treatment of Rejection.(which is still debatable).
Be Prepared to Stop: Limitations:
MFI cutoff values are different from one center to another and also according o transplantation program, Many studies reveled that low MFI associated with poor graft outcome and vice versa, and many laboratory factors affected on the result of MFI and keep it dynamic more and more and difficult to relay on it to interprets with clinical practice.
Rerouting: New Directions:
Some antibody react with more than antigens which share together(which mainly epitope that share antigenicity in more than HLA antigen) and so we should change antigens bead to epitopes.
Bumps in the Road: Conformation—An Integral Component to Antibody Pathogenicity?
The degree and strength of antibody binding is influenced by antigen conformation , e.g. an antibody to Bw6 was identified on class I SAB with MFI values >17 000 except for the B*46:01- and B*73:01-bearing beads, which had MFI values of ~2000 and ~1500, respectively, which might be depend on sequence of amino acids(eplet) which different from site in epitopes to another which change antigenicity.
The difference at this position infers a change in epitope conformation that diminishes antibody affinity for the target antigen , all these can affect MFI value.
Divided Highway: Antigens Versus Antibodies:
Antigens might be expressed in vivo more than invitro which induced antibody response so DSA with low MFI might be not reflect the antigen expression in vivo so its difficult to relay on MFI for clinical outcome.
Merge: “Functional” Methods to Assess Antibody Pathogenicity:
Complement fixing characteristics of HLA antibodies, by adding (c1q, c3d or c4d) as a surrogate for complement system in vivo many studies revealed its association with poor graft outcome and other not .but sometimes ABMR occurred due to non-complement dependent pathway.
Guides to Antibody Assessment:
So antibody assessment needs more and more technical advances and innovations , but still we should work as a multi -disciplinary team and deal case by case assessment to guide antibody with each case.
To deal with the total view clinical and technical aspects.
☆Complement-Binding Anti-HLA Antibodies
and Kidney-Allograft Survival
_____________________________________________
Summary:
▪︎In this study the authors hypothesized that the complement binding properties of donor-specific anti-HLA antibodies detected after transplantation are involved in kidney-allograft failure. So, they conducted this study to:
1) Define the full spectrum of kidney-allograft injury according to the C1q-binding properties of donor-specific anti-HLA antibodies in a large population-based study.
2) Determine whether assessment for the presence of C1q-binding donor-specific anti-HLA antibodies after transplantation might improve risk stratification for kidney-allograft loss.
Methods:
__________
▪︎ Study population : Patients who received kidney allografts at two transplantation centers in Paris between January 1, 2005, and January 1, 2011. Patients were followed until April 15, 2012. An external-validation cohort was also included.
▪︎All transplants were compatible with the ABO blood group.
▪︎ A negative result of cross-matching for IgG T-cell and B-cell complement-dependent cytotoxicity was required for all recipients.
Clinical Data:
______________
▪︎ Anonymized data from registries were entered on day 0 and 6 months and 1 year after transplantation for each patient and were updated annually thereafter.
▪︎ The derivation-cohort data and validation-cohort data were obtained.
▪︎All cases of acute clinical rejection (defined by deterioration in graft function, proteinuria, or impaired renal function and histopathological evidence of rejection, according to the consensus rules of the international Banff classification criteria), were documented.
◇Histologic and Immunochemical Tests:
____________________________________________
▪︎ Biopsies were performed in patients with/without acute clinical rejection episodes diagnosed in the first year of transplantation and were scored and graded from 0 to 3 according to the updated Banff criteria with respect to the presence of donor-specific anti-HLA antibodies, C1q-binding status, and clinical course.
▪︎ C4d staining was performed
◇Detection and Characterization of Donor-Specific Antibodies:
______________________
▪︎All patients were tested for the presence of circulating donor-specific anti-HLA on Luminex platform.
▪︎ Serum samples from patients with circulating donor-specific anti-HLA antibodies were analyzed for the presence of C1q-binding donor-specific anti-HLA antibodies with the use of single-antigen
flow bead assays.
♧ Discussion:
_______________
▪︎The presence of complement-binding donor-specific anti-HLA antibodies detected in the first year after transplantation was an independent predictor of kidney-allograft loss more than 5 years after transplantation and significantly improved individual risk stratification for graft failure.
▪︎Patients with complement-binding donor-specific anti-HLA antibodies after transplantation had a graft injury phenotype characterized by microvascular inflammation and complement split-product C4d deposition.
▪︎ C1q-binding donor-specific anti-HLA antibodies remained strongly associated with the risk of graft loss regardless of the mean fluorescence intensity of the antibodies.
▪︎ C1q testing may help identify patients at risk, despite C4d negativity.
▪︎Assessment for complement-binding donor-specific anti-HLA antibodies may provide an early indication of the potential for complement-mediated injury, without the functional requirement for progression through the pathway to C4d deposition.
▪︎ Detection of complement-binding donor-specific anti-HLA antibodies may indicate which of the antibodies present have the capacity to activate the complement cascade.
☆This study may provide a basis for future clinical trials (e.g., a C5 inhibitor [eculizumab] or a C1 inhibitor)
☆The level of evidence of this study:
____________________________________
Level II
☆The type of this study:
________________________
Population based large cohort study
What this type of this study?
This is a cohort study
What is the level of evidence demonstrated by this study?
Level 2 of evidence.
Summary of the article
Complement-Binding Anti-HLA Antibodies and Kidney-Allograft Survival
This article discusses the impact of the anti HLA antibodies on the kidney graft survival. Although anti-HLA antibodies are considered to
be harmful, there is a wide spectrum of graft injury related to these antibodies, ranging from no recognizable damage to florid rejection.11,12 Such
a varied effect underscores the need to define distinct graft phenotypes and outcomes according to the presence or absence and characteristics of donor-specific anti-HLA antibodies after transplantation.
Methods
We enrolled all consecutive patients who underwent kidney transplantation at Necker Hospital and Saint-Louis Hospital (Paris) between January 1,
2005, and January 1, 2011, in this populationbased study. Patients were followed until April 15, 2012.
We also included an external-validation cohort comprising patients who underwent kidney transplantation at Foch Hospital (Suresnes, France) between January 1, 2004, and January 31, 2010
The transplantation allocation system was identical for the three centers and followed the rules of the French national agency for organ procurement (Agence de la Biomédecine)
All transplants were compatible with the ABO blood group.A negative result of cross-matching for IgG T-cell and B-cell complement-dependent cytotoxicity was required for all recipients
Clinical Data
Clinical data on the donors and recipients in the derivation cohort and the validation cohort were obtained from two national registries. Anonymized data from these registries are prospectively entered at specific time points for each patient (on day 0 and 6 months and 1 year after transplantation) and are updated annually thereafter)
The derivation-cohort data were obtained from the database on April 15, 2012, whereas the validation-cohort data were obtained on December 19, 2012.
We documented all cases of acute clinical rejection, defined by deterioration in graft function, proteinuria, or impaired function and histopathological evidence of rejection, according to the consensus rules of the international Banff classification criteria.
Immunosuppression protocols and treatment of allograft-rejection episodes after transplantation were similar among the centers.
Histologic and Immunochemical Tests
We used specimens from protocol-specified graft biopsies performed 1 year after transplantation in 845 patients without any acute clinical rejection episodes diagnosed in the first year after transplantation, as well as specimens from biopsies
performed in 171 patients with acute allograft rejection during the first year after transplantation.
All graft-biopsy specimens were scored and graded from 0 to 3 according to the updated Banff criteria by three trained pathologists who were unaware of the patient’s status with respect to the presence of donor-specific anti-HLA antibodies,
C1q-binding status, and clinical course.
C4d staining was performed by means of immunochemical analysis on paraffin sections with the use of polyclonal human anti-C4d antibodies.
Detection and Characterization of Donor-Specific Antibodies
All patients were tested for the presence of circulating donor-specific anti-HLA antibodies in banked serum samples.
donor-specific anti-HLA antibodies were analyzed in a blinded fashion.
Statistical Analysis
We used means and standard deviations for the description of continuous variables, with the exception of mean fluorescence intensity, for which we used the mean and standard error. We compared means and proportions using Student’s t-test and the chi-square test. Survival was analyzed from the time of transplantation to a maximum of 7 years, with kidney-graft loss as the event of interest
Results
Baseline Characteristics of the KidneyAllograft Recipients
In total, 1016 patients undergoing renal transplantation (695 at Necker Hospital and 321 at SaintLouis Hospital) were included in the main analysis.
Three distinct populations were identified after transplantation, according to the presence or absence of donor-specific anti-HLA antibodies and complement-binding capacity.
– 700 patients without circulating donor-specific anti-HLA antibodies
– 239 patients with non–complement-binding donor-specific anti-HLA antibodies,
– 77 patients with complement-binding donor-specific anti-HLA antibodies
Kidney-Allograft Injury
In the first year after transplantation, acute clinical rejection developed in 171 patients:
– 96 patients had T-cell–mediated rejection (56%)
– 75 had antibody-mediated rejection (44%).
T-cell– mediated rejection occurred in
– 14 of 77 patients with donor-specific anti-HLA antibodies plus C1q-binding capacity (18%),
– in 30 of 239 patients with donor-specific anti-HLA antibodies without
C1q-binding capacity (13%)
– in 52 of 700 patients without donor-specific anti-HLA antibodies (7%)
Antibody-mediated rejection occurred in
– 37 patients with C1q-binding donorspecific anti-HLA antibodies (48%)
– 38 patients with non–C1q-binding donor-specific anti-HLA antibodies (16%)
Kidney-Allograft Survival
The median follow-up after transplantation was 4.8 years. The risk of graft loss
according to the donor-specific anti-HLA antibodies–C1q status at day 0 and the status after transplantation revealed that patients with C1qbinding donor-specific anti-HLA antibodies after transplantation had the highest risk of graft loss.
The HLA (second paper)
Searching for a new method to Identify clinically relevant HLA antibodies of clinical importance was a challenging
The Flow PRA is a screening test to detect HLA antibodies qualitively
while SAB assays is a semiquantitative test detecting the antibody specificity.
These two tests are sensitive and specific .
Luminex was specific and sensitive in detecting HLA antibodies.
mean fluorescence intensity (MFI) is a numerical value reported from SAB results .
It reflects a certain bead’s relative fluorescence which is affected by many variables.
The drawbacks
-If the threshold is low, false-positives may prevent a patient from receiving a compatible graft ;and if high, there may be increased risk that a patient will have AMR.
-Most studies demonstrated that low MFI levels correlated with favorable outcome and high indicated poor graft outcome but the range of MFI values is not standardized.
-Also SAB results are variable between different labs
-For MFI values stems from interferences due to antibody-dependent complement activation. Complement split products bind to antigen–antibody complexes on the beads blocking binding sites for secondary antibodies
-Low MFI DSAs associated with poor outcomes can be explained by competing of IgM HLA antibodies, interfering with the binding of IgG HLA antibodies
– False-positive result can occur due to antibody binding to denatured antigens.
-On the other hand high-MFI DSAs were not associated with poor graft outcome this can be attributed to denatured proteins .
– It is unknown if the reactivity to native and denatured HLA antigens is hazardous or not
Cross-reactive groups (CREGs) described the serologic phenomenon of multiple specific HLA antigens that reacted with a single antibody.
Therefore the antibody reactivity is directed to the epitope rather than to individual HLA antigens, known before as non-DSAs, now considered as DSAs.
The degree of antibody binding is affected by antigen formation.
The current version of HLAMatch-maker and the epitope registry factor (eplets) that have recently become a major component of compatibility determination in many laboratories.
Difference in MFI may be due to effect of more than one of the amino acid differences.
DSA that binds a bead in vitro may not bind to the corresponding antigen in vivo due to low expression .
On the contrary Wiebe et al, stated that the de novo antibody produced post transplantation are directed to HLA-DQ antigens despite low expression of the DQ loci .
The harmful effect of an antibody cannot not be assessed based solely on MFI levels.
Some studies demonstrated significant correlation between complement fixation and poor allograft outcome while others did not.
Meanwhile complement-independent AMR can occur .
HLA antibody detection and identification is subjected to further research
DSA carry negative impact on graft survival but need to know more about DSA MFI, anti HLA class 1,antiHLA class 2,anti A,B,DR, DQDP, IG g subclass ,preformed or post transplant
IN THIS ARTICLE AIM OF STUDY TO COMPARE CPLEMENT FIXING FROM NON COMLEMENT FIXING DSA ON allograft survival .
1016 patient enrolled in this study from NECKER HOSPITAL and Saint -LOUIS HOSPITAL (PARIS) with external validation from FOCH-HOSPITAL
Period from 2005 to 2011.
845 underwent protocol biopsy 1 year after transplantation .
171 for cause biopsy (graft rejection).
DSA measured at 0 and at one year post transplantation .
C1Q binding status was assessed.
5 year graft survival in patient with complement fixing DSA was inferior to those who has non complement fixing DSA .
Risk stratification of patient change if we know that DSA complement fixing from non complement fixing but from
my point of view apart from risk assessment and prognostic information need to know if we need to do C1Q status to All DSA positive patient ,
and if there is any change in desensitizing regimen or line of management of Ab mediated rejection between C1Q positive or negative patient ?,
what about cost effectiveness?.
What this type of this study?
This is cohort study
What is the level of evidence demonstrated by this study?
Level 2 of evidence .
The effect of anti-HLA antibodies post transplant varies from unrecognized damage to florid rejection according to the characteristics of anti-HLA antibodies.
The capacity of anti-HLA antibodies to bind complement and activate the classic complement cascade determines the cytotoxicity of these antibodies.
This study aimed to determine whether the assessment of presence of C1q binding DSA will improve risk stratification for graft loss.
A prospective cohort study included 1016 kidney transplant recipients with compatible ABO blood group and negative CDC crossmatch, they were followed up for 4.8 years.
It evaluated the immunologic characteristics before and after transplantation including graft phenotype.
Acute rejection was defined by deterioration of graft function, proteinuria and histopathological evidence of rejection.
Patients were tested for DSA at time of transplantation, one year post transplant and during episode of acute rejection.
Sera of patients with DSA were analyzed for presence of C1q binding DSAs.
Patients were identified according to absence or presence of DSA and complement binding capacity.
Detection of complement binding DSA in first year post transplant was an independent predictor of graft loss 5 years post transplant and significantly improved risk stratification for graft failure.
Patients with complement binding DSA had graft injury characterized by microvascular inflammation and C4d deposition.
C1q binding DSA was strongly associated with the risk of graft loss regardless the MFI of antibodies.
Assessment for complement binding DSA may indicate the potential of complement mediated injury before progression through the pathway to C4d deposition.
Identifying complement binding ability may be important as new therapeutic agents as C5 inhibitor (eculizumab) and C1 inhibitor targeting complement are increasingly used in kidney transplant recipients.
Limitations:
It didn’t investigate the changes in capacity of DSA to bind complement and the effect of treatment on these antibodies.
prospective cohort study
Level of evidence II
Please give a summary of this article
Two transplant centers in Paris were evaluated over a period of six years, which monitored the circulation of HLA antibodies and complement-binding capacity after transplantation and its role in graft failure.
All patients had ABO compatibility and negative results for both crossmatch and complement-dependent cytotoxicity. SAB Luminex collected on day 0 and 365 dosing HLA A, B, Cw, DR, DQ, DP and C1q binding associated with biopsies with evidence of clinical and laboratory rejection or after a period of one year.
1016 patients were selected, 845 without symptoms and 171 with TCMR (56%) and AMR (44%).
When stratifying the data, the presence of C1q+ showed a higher risk of losing the graft and showing lower levels of Creatinine Clearence. Histopathological findings showed microvascular infiltration (87%), interstitial tubular inflammation (36%), endarteritis (23% and transplant glomerulopathy (22%).
The presence of c1q complement binding after transplantation showed a Harzard ratio of 4.48 independent of the positivity of other DSAs.
The presence of C1q+ is an independent factor of graft loss and independent of MFI values, only confirming the need to expand the investigation of ASD beyond HLA standards, in addition to more sensitive and specific methods.
What is the level of evidence demonstrated by this study?
What this type of this study?
Cohort study – IIb
Prospective population based study carried out in France between 2005 through 2012.
Aim: To assess the correlation of complement binding DSAs with allograft survival and post transplantation complications in comparison to patients with non complement binding DSAs and patients with negative DSAs.
Method:
1016 patients were enrolled in this prospective study.HLA A,B,Cw and DR,DQ and DP were assessed by Luminex
All the patients were tested for DSAs at the time of transplantation and one year post transplantation.
Protocol allograft biopsy was performed one year post transplantation or at any time during the first year if any patient developed acute rejection or allograft dysfunction,looking for changes related to rejection process according to Banff classification.
C1q binding test was performed in all patients with circulating DSAs to categorize into complement fixing and non complement fixing DSAs.
Results:
3 distinct populations identifed :
700 patients with negative DSAs.
239 patients with DSAs non complement fixing.
77 patients found to have complement fixing DSAs.
171 patients developed acute rejection in the first year post transplantation, of which 96 had CMR and 75 had AMR.
Of the 96 patients with CMR :
14 with C1q positive DSAs patients.
30 C1q negative DSA
52 DSA negative patients.
Of the 75 patients with AMR:
37 patient with C1q positive DSAs
38 patients with C1q negative DSAs.
Of the 77 patients with c1q positive DSAs 37 (48%)developed AMR.
Of the 239 patients with c1q negative DSAs 38(16%) developed AMR.
This result revealed strong evidence that c1q positive DSAs are associated with higher incidence of AMR in comparison to C1q negative DSAs patients and those patients with negative DSAs(48% vs16%) statistically significant with p value of less than 0.001.
Further more ,c1q positive DSAs patients showed more extensive micro vascular inflammation and transplant glomerulopathy and higher scores for graft peritubular c4d deposition than for both C1q negative DSAs and negative DSAs patients.stastically significant.
Gfr of the c1q patient was significantly lesser than c1q negative DSAs and patients with negative DSAs over median follow up period of 4.8 years.
Similarly 5 years graft survival was 54% in c1q positive DSAs patients vs 93% and 99 % for those with c1q negative DSAs and DSAs negative patients respectively.statistically significant with P value of less than0.001.
An important observation was made by this study, that is MFI level, as it was non significant risk factor in those patients who are c1q positive DSAs (same risk in those with MFI above or below 6000)
Conclusion:
Complement fixing DSAs are associated with higher risk of AMR and bad long term outcome.
It’s representing an important suttogate marker for evaluation and risk stratification for kidney transplant recipients.