2. You have seen a 53-year-old CKD patient on HD due to IgA nephropathy who is fit for transplantation (all work up is acceptable). His son, 111 mismatch ABO compatible is willing to donate a kidney. FCXM is negative but DSA was identified (CW15 with MFI 1300)
- What are the techniques used to detect DSA?
- How you manage his transplantation?
- What is the relevance of CW15 with MFI 1300?
Dear All
Thank you very much for your input.
SAB is the technique of choice, remember that CDC is inaccurate. Regarding the clinical significance of HLA C DSA, it is very controversial. In my opinion, it would be relevant at a high MFI level (>5000 and in case of sensitisation such as the previous transplant).
We transplanted a few patients like this. The only trouble I had, was a patient with HLA C DSA with MFI 5500. This kidney did not last for long, looks behaved like anti-class II antibodies (indolent, more chronic AMR).
I strongly suggest standard immunosuppression with basiliximab induction and careful monitoring of the graft function and the DSA.
1.Luminex-SAB is the most accurate method for DSA.
2.Induction with basiliximab, methylprednisolone and then triple therapy with CNI, MMF and prednisolone
3.HLA CW15 with low MFI shows low immunological risk
1 . single antigen bead by luminex
CDC
flow cytometery
2, proceed for transplantation with standard immunosuppression
Basiliximab and maintenance ( steroid , Tac ,MMF)
3.the relevance of CW15 with MFI 1300
low risk for rejection , problems occurs with titre more than 5000
1) What are the techniques used to detect DSA?
Single antigen bead.
CDC
Flowcytometry
2)How you manage his transplantation?
Induction :Basiliximab.
Maintenance : Tacrolimus , Mycophenolate, Steroid.
Close DSA and renal profile monitoring.
3)What is the relevance of CW15 with MFI 1300?
low risk for antibody mediated rejection post transplant.
CDC cross matching
flow cytometry cross matching
Sequence-specific oligonucleotide probes (SSOP) typing
Induction therapy: using Basiliximab
maintatnce therapy: Tacrolimus, Mycophenolate mofetil, corticosteroid.
This level may be considered low risk as MFI > 3000 is the level confer higher risk of rejection
Techniques to identify DSA
Management
CW15, MFI 1300
Gold standard method is SAB (Luminex) as it is more sensitive, specific and accurate than other methods like CDC and FCXM.
This a standard risk mismatch, so I will opt for the following IS regimen:
Induction: Basiliximab
Maintenance: Tac, MMF and steroids
Few studies were looking for the outcome of renal transplant patients with anti-HLA CW DSA, it was concluded that anti HLA-C DSA increase the risk of AMR and lower graft survival. When compared to anti-HLA- A, B, DR and DQ antibodies, they were associated with similar incidence of AMR and same graft survival.
In our discussed case scenario, anti HLA CW15 MFI 1300 is low (above 3000 would be highly significant, so he doesn’t need desensitization and transplantation can be done.
Q1- CDC crossmatch
Flowcytometry
SAB luminex
Q2- Induction with basiliximab or methylprednisolone followed by tacrolimus, MMF and steroid.
Follow up of the antibody titer.
Q3- Not significant
There is CDC method ( serological method ) which is not accurate to detect DSA.another method is the solid phase, which consists of 2 methods of flow cytometry( DSA against B cells and /or T cells & Luminex which can detect the specificity of DSA , called a single antigen (SAB) which is the most accurate for detecting DSA .
This patient has a stander risk for transplantation as ABO compatible 111 mismatch, FCXM was negative & CW15 with low MFI 1300
. induction with basiliximab , & stander maintenance immunosuppressive meds .
class I DSA , with low MFI, it is considered as low immunological risk, needs follow up post-transplant if rejection occurs, to do kidney biopsy & send for the level of SAB.
Techniques used in DSA detection
Solid phase Ab which includes ELISA and bead technology (luminex) which more commonly used especially the last one.
Flow crossmatch more sensitive than CDC
CDC the basic and functional test
The management of this transplant from son to father with ABO compatible,3 mismatch,negative FCXM and 1300MFI DSA Ab against CW15( which considered not high ) so can manage him as low to standard immunological risk treated with basiliximab 2 doses and keep him on triple immunosuppressants .
HLA- C matching can give better result in kidney transplant and from immunological point HLA- C can induce antibody response and presents peptides as HLA- A,B and DR molecules so it may be as important as other loci.
Reference
1- A K, A M, A S, M EK, A H.An update on crossmatch techniques in transplantation [ASNRT fellowship in Clinical Transplantation]2017;03(04).available from: http: //dx.doi.org/10.4172/2472-1220.1000160
2- Torpey N, Moghel NE, Waston E, Tolbot D, editors. Renal Transplantation. London,England: Oxford University press ;2010
3- Pratschke J, Dragun D, Haue IA, Horn S, Mueller TF, Schemmer P , et al .Immunological risk assessment: The key to individualised immunosuppression after kidney transplantation. Transplant Rev(Orlando)[Internet].2016;30(2);77-84. Available from;http: //dx.doi.org/10.1016/j.trre.2016.02.002
4- Frohn C, Fricke L, Puchta JC, Kircher H.The effect of HLA- C matching on acute renal transplant rejection. Nephrol Dial Transplant [ASNRT fellowship in clinical Transplantation].2001;16(2):355-60. Available from;http: //dx.doi.org/10.1093/ndt/16.2.355
we can proceed this transplantation with standered immunosuppression with induction by basiliximab and triple immunosuppression with steroid , MMF and tacrolimus and monitor DSA level post-transplant
Not relavent as MFI is less than 5000
Techniques to detect DSA?
Serological: less accurate
Solid Phase assay- More sensitive and reproducible
· ELISA Variation is possible among kits
· Flow Cytometry- Highly sensitive detecting ABs towards T and B cells
· SAB- Luminex Single antigen Beads- Most commonly used with possible missing of non HLA antibodies
Management of this transplantation
Both donor and recipient are compatible regarding ABO with HLA mismatch 1.1.1,negative FCXM , positive DSA to CW15 with MFI 1300. Standard immunosuppression is needed. Also, DSA monitoring and protocol biopsy to look for AMR
Relevance of CW15 with MFI 1300
MFI of 1300 is low ; significance needs 5000 plus. studies have shown deleterious impact of CW15 mismatch on graft survival.
Referrence:
C Frohn et al. The effects of HLA C on acute renal transplant rejection. Nephro Dial transplant. 2001 Feb;16(2):355-60.
1-Cytotoxic (cell-based) antibody screening
The method is similar to that of serologic typing . the patients is mixed with donor lymphocytes plus complement plus vital dye . positive test lead to cell lysis and dye uptake .
2- Solid phase antibody screening
This method employs soluble or recombinant HLA molecules but not lymphocytes .
2-A Enzyme-linked immunosorbent assay platform:
In this method, purified HLA molecules are applied to enzyme-linked immunosorbent assay (ELISA) platforms and will bind individually to HLA antibody after the addition of recipient serum . Enzyme conjugated antibodies to IgG (human) is then added to detect the presence of HLA antibody in the serum which is bound to the antigen. Detection is performed by optical density reading.
2-B Microbead platform/single-antigen beads:
Pooled panel beads with several different class Ⅰ or Ⅱ HLA antigens on a bead yield a positive or negative result and are utilised for screening . The phenotype or also known as ID beads are individually coated with class Ⅰ or Ⅱ HLA antigens of an individual patient-derived cell line. Microbead that is fluorescent dye conjugated is then added to detect the presence of HLA antibody in the serum which is bound to the antigen. Fluorescence detection can be done traditionally using
a flow cytometer (Flow PRA®) or
via the single-antigen beads (SAB) Luminex® platform.
===================================================
How you manage his transplantation?
This patient has living related donor, ABO COMPATIBLE ,WITH 50% mismatch . FC XM IS NEGATIVE . But low titre (MFI 1300) positive CXM FOR CW15 .
I consider this patient as alow to moderate risk I will manage this patient by
Induction using ATG plus triple is medication (MMF ,PROGRAF, PREDNISOLONE )
With monitoring patient by graft function, protein urea and DSA monitoring .
===========================================================
What is the relevance of CW15 with MFI 1300?
Generally HLA CW antigens characterized by lower degree of expression, low immunogenicity.
Therefore in this case with this low titre CW DSA is of low risk for rejection . although the risk of rejection is still there ,means that DSA MONITORING IS INDICATED .
================================================================
References:
1) Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and crossmatch: A comprehensive review. World J Transplant. 2017 Dec 24;7(6):339-348. doi: 10.5500/wjt.v7.i6.339. PMID: 29312863; PMCID: PMC5743871.
2) Santos S., Malherio J., Tafulo S., et al. Impact of preformed donor-specific antibodies against HLA class I on kidney graft outcome: Comparative analysis of exclusive anti Cw vs anti-A and /or -B antibodies. World J Transplant , 2016;6(4):689-696.
Q1: Techniques to detect DSA:
Q2: How you manage his transplantation?
Ref: Patel AM, Pancoska C, Mulgaonkar S, Weng FL. Renal transplantation in patients with pre-transplant donor-specific antibodies and negative flow cytometry crossmatches. Am J Transplant 2007; 7:2371
Q3: Relevance of CW15 with MFI 1300?
Ref: Donor-Specific Antibodies in Kidney Transplant Recipients Rubin Zhang
CJASN Jan 2018, 13 (1) 182-192; DOI: 10.2215/CJN.00700117
1. Techniques used to detect DSA are Cell based (cytotoxic) and Solid phase antibody screening *ELISA
*Single antigen bead technology ( very sensitive technique) using Luminex method or Flowcytometry
2. Start with Induction immunosuppression with ATG and triple immunosuppression tacrolimus , MMF and steroids
With strict Follow up KFTS , A/C ratio , DSA and Renal biopsy if needed
3. DSA aganist HLA C causes high incidence of Antibody mediated rejection and Graft dysfunction
But still MFI is less than 5000 so low risk for AMR
single anti body detection is usualy done by singe bead (luminex)
this patient is low risk so could be either low dose ATG or preferably basiliximab
CW15 with MFI 1300 is low so may not bee accepted as high risk
DSA can be detected by-
1.CDC method(serological)
2.solid phase-flow cytometry
-ELISA
-SAB
This tx can be managed with normal risk tx,with
ATG(3mg/kg)
Tacrolimus 0.10mg/kg with target level of 10
MMF 360mg tds
Corticosteroid.
CW15 with 1300 mfi not relevant as it less than 5000 single ag and less than 10000 combining two ag.
techniques used to detect DSA
a. Single Antigen Beads (SAB), Luminex: it’s now the technique of choice.
b. complement dependent cytotoxicity (CDC) : inaccurate giving false data.
c. FLOW CYTOMETRY
How you manage his transplantation?
FCXM is negative, DSA was identified (CW15 with MFI 1300) WHICH IS NOT SO MUCH HIGH,AS MENTIONED ALL OTHER LAB IS OK I WILL RECOMMEND TO PROCEED WITH TRANX WITH INDUCTION BY ATG OR BASILIXMAB PLUS METHYLEPRED WITH MAINTENACE WITH TACRLOLIMUS MMF CCS.What is the relevance of CW15 with MFI 1300?NO SERIOUS THING AS PATIENT HAS LOW MFI 1300 NOT EXCEEDING 5000.( ONLY SERIAL DSA ASSAY POST TRANSX )
techniques used to detect DSA
a. Single Antigen Beads (SAB), Luminex: it’s now the technique of choice.
b. complement dependent cytotoxicity (CDC) : inaccurate giving false data.
c. FLOW CYTOMETRY
How you manage his transplantation?
FCXM is negative, DSA was identified (CW15 with MFI 1300) WHICH IS NOT SO MUCH HIGH,AS MENTIONED ALL OTHER LAB IS OK I WILL RECOMMEND TO PROCEED WITH TRANX WITH INDUCTION BY ATG OR BASILIXMAB PLUS METHYLEPRED WITH MAINTENACE WITH TACRLOLIMUS MMF CCSWhat is the relevance of CW15 with MFI 1300?NO SERIOUS THING AS PATIENT HAS LOW MFI 1300 NOT EXCEEDING 5000.( ONLY SERIAL DSA ASSAY POST TRANSX )
What are the techniques used to detect DSA?
Cell based immunoassay(CDC, flow cytometry ).
solid phase immunoassay ( ELISA, Luminex SAB).
How you manage his transplantation?
risk stratification need to be assessed before management protocol ;
patient with anti HLA C less immunogenic than anti HLA A,B DR and low titer (1300) but there is risk since It is DSA so patient between standard to moderate risk .
I think he need ATG based regimen but low total dose (3gm) with TAC.MMF and steroid with post transplant DSA monitoring .
What is the relevance of CW15 with MFI 1300?
CW15 recently has deleterious effect on graft survival compare with previous study.
1) for DSA detection : 1* Complement Dependant Cytotoxicity (CDC)
2* Solid phase Assay : best is Single Antigen Bead ( SAB ) –
Luminex
2) having no apparent previous sensitizing events , with low MFI for his DSA : induction with ATG then to be on Triple therapy ( steroid ,CNI,antiproliferative ) , regular DSA monitoring post transplantation
3) not relavent as MFI is less than 2000
1. What are the techniques used to detect DSA?
2. How you manage his transplantation?
3. What is the relevance of CW15 with MFI 1300?
Can use: AHG-CDC, FCMX and specifics methods ( solid-phase immunoassay ELISA or LUMINEX)
I would use a protocol with strong induction (Thymoglobulin) and maintenance with corticosteroids + ICN + MMF.
Patient already sensitized (pregnancie), with identification of ASD and risk of autoantibodies and HLA Typing mismatch.
There´s no relevance, because de MFI < 5000.
*** What are the techniques used to detect DSA?
••• Cyrtotoxic (cell-based) antibody screening:
Where 30-40 donor lymphocytes mixed with recipients serum in individual wells along with complement and dye.
Where the serum contains antibodies that bind to the cell surface with adequate density complement pathways are activated which results in cell death and uptake of the dye . The degree of cytotoxicity is expressed as percentage PRA (panel reactive antibody). It is a tool that can be employed to approximate the risk of a given
recipient of having a positive crossmatch. This is to a likely organ donor taken from a similar population.
•••Solid phase antibody screening :
>>> Enzyme-linked immunosorbent assay plateform :
In this method, purified HLA molecules are applied to enzyme-linked immunosorbent assay (ELISA) platforms and will bind individually to HLA antibody after the addition of recipient serum. Enzyme conjugated antibodies to IgG (human) is then added to detect the presence of HLA antibody in the serum which is bound to the antigen. Detection is performed by optical density reading.
>>>Microbead plateform / single -antigen beads:
Pooled panel beads with several different class Ⅰ or Ⅱ HLA antigens on a bead yield a positive or negative result and are utilised for screening .The phenotype or also known as ID beads are individually coated with class Ⅰ or Ⅱ HLA antigens of an individual patient-derived cell line. Microbead that is fluorescent dye conjugated
is then added to detect the presence of HLA antibody in the serum which is bound to the antigen. Fluorescence detection can be done traditionally using a flow cytometer or via the single-antigen beads (SAB) Luminex platform. These estimate PRA by the proportion of positive beads. SAB are individually coated
with a single HLA antigen and yield a list of distinct
antibody specificities .Specificities are subsequently compared with HLA frequencies in the donor population to determine the calculated panel-reactive antibody . This yields the best estimate of the likelihood of a positive crossmatch/donor specific antibody to a randomly selected donor.
How you manage his transplantation?
This patient is ABO compatible with 111 mismatch
FCXM is negative with DSA positive ( CW15) with MFI 1300 , the patient has low cutoff MFI , we can proceed this transplantation with standered immunosuppression with induction by basiliximab and triple immunosuppression with steroid , MMF and tacrolimus and monitor DSA level post-transplant.
*** What is the relevance of CW15 with MFI 1300?
Patients with high level pre-transplant HLA C DSA MFI more than 3000 or history of previous transplantation have increasing in risk of AMR in one year post transplant with poor graft survival but this patient has low level of DSA with cutoff 1300 so there is decrease risk of AMR in one year post-transplantation.
References:
1.PROF: AHMED HALAWA lecture
2.Pei R, Wang G, Tarsitani C, Rojo S, Chen T, Takemura S, Liu A, Lee J. Simultaneous HLA Class I and Class II antibodies screening
with flow cytometry. Hum Immunol 1998; 59: 313-322 [PMID: 9619770 DOI: 10.1016/S0198-8859(98)00020-2].
3.Pei R, Lee JH, Shih NJ, Chen M, Terasaki PI. Single human leukocyte antigen flow cytometry beads for accurate identification of human leukocyte antigen antibody specificities. Transplantation 2003; 75: 43-49 [PMID: 12544869 DOI
10.1097/01.tp.0000040431.80510.98]
4.Zachary AA, Braun WE. Calculation of a predictive value for transplantation. Transplantation 1985; 39: 316-318 [PMID:
3975995 DOI: 10.1097/00007890-198503000
00024.
One of the following techniques
– Serological
– Solid-phase assay (more sensitive than the serological and more reproducible)
1. ELISA
Variations between the kits
2. Flow cytometry
Using micro-particles, each coated with soluble single HLA antigen, to detect and measure the channel shift associated with antibody binding to the beads.
3. Luminex® Single Antigen Beads (SAB)
Commonly used
How do you manage his transplantation?
Basiliximab induction & triple immunosuppression, which include:
Prednisolone, MMF, &TAC
DSA with MFI values above 5000 were considered positive, but this titer in this patient is still at low-risk ABMR
Techniques to detect DSA are-
1) CDC – advantage of being easily available, low cost, more specific for complement fixing antibody , but have disadvantages of being less accurate having both false +ve in presence of autoantibodies( IgM), certain drugs like INH/ Hydralazine and False negatives- in presence of non complement fixing antibodies, even low titre of DSA etc
2) FLOW CYTOMETRY CROSSMATCH- pros of being more sensitive then CDC and can detect non complement fixing antibodies, cons of having false +ve results due to presence of non HLA/ non specific antibodies like after exposure of Rituximab. It has low specificity also
3) LUMINAX SAB/ VIRTUAL CROSS MATCH- most sensitive and highly specific in identifying DSA, detect both complete fixing and non fixing HLA antibodies, neither detect non HLA antibody nor non specific IgM antibodies, largely replaced cross match of choice with flow cytometry in hospital now. Only problem is very high cost.
How to manage this transplantation?
Related( son) being donor , ABO compatible with negative flow cytometry cross match and positive HLA C DSA with low titer of MFI 1300, i would like to go with basiliximab induction and triple immunosuppressive regimen with prednisolone, tacrolimus and MMF.
Post transplantation this patient needs to be more closely monitored.
What is the relevance of CW15 with MFI 1300?
HLA class C DSA presence is of less clinical significance but not totally benign and with high titre definitely it become high risk transplantation. Transplantation can be done in such patients if titre is low, with optimisation of immunosuppresive as per the risk and definitely more close monitoring of such patients post Tx with aggressive investigation and treatment if any biochemical or clinical or immunological parameter deteriorates.
What are the techniques used to detect DSA?
one of the following techniques
– Serological
– Solid phase assay (more sensitive than the serological and more reproducible)
1. ELISA
Variations between the kits
2. Flow cytometry
Using micro-particles, each coated with soluble single HLA antigen to detect and measure the channel shift associated with antibody binding to the beads.
3. Luminex® Single Antigen Beads (SAB)
Commonly used
How you manage his transplantation?
Induction by Standard immunosuppressant
and maintenance by triple IS drugs with careful monitoring by protocol biopsy
What is the relevance of CW15 with MFI 1300?
Classically anti-HLA-Cw are considered less immunogenic and are not considered in many organ allocation systems or immunologic risk stratification algorithms, including in Portugal. However, data from literature con- firms that their presence is as deleterious as DSA anti-HLA A/B/DR/DQ.
Thus we should take HLA-C typing and respective antibody identification into account in sensitized patients, in order to access risk stratification and establish the need for correct induction or desensitization therapies.
Key-Words: Donor-specific antibodies (DSA), anti-HLA-Cw antibodies, antibody-mediated rejection (AMR).
Reference:
Kidney transplantation in patients with preformed and exclusively anti-HLA-Cw donor specific antibody
Sofia Santos1, Ana Castro1, Andreia Campos1, Sofia Pedroso1, Leonídio Dias1, Castro Henriques1
Serviço de Nefrologia e Transplantação Renal, Centro Hospitalar do Porto, Porto, PORTUGAL.
Received for publication: Nov 11, 2016 Accepted in revised form: Jan 30, 2017
Con.to reference:
Dr Ahmed Halawa lecture
What are the techniques used to detect DSA?
These include;
Serological– Old technique and less accurate
Solid Phase Essay- More sensitive and reproducible
· ELISA- Variation occurs between kits
· Flow Cytometry- Highly sensitive and detects antibodies to T and B cells
· SAB- Luminex Single antigen Beads- Most commonly used but may miss non HLA antibodies
How you manage his transplantation?
The donor and recipient are ABO compatible and have HLA mismatch 1.1.1. FCXM is negative. DSA to CW15 is positive and MFI is 1300. I will go for induction with ATG and maintenance with Tacrolimus, MMF and prednisolone . Follwo up with DSA monitoring and biopsy as per protocol to look for AMR
What is the relevance of CW15 with MFI 1300?
MFI is low but still graft outcome may be variable. CW15 historical thought to be low risk but recent studies have have shown signifiicant relation with AMR. Native anti HLA Cw havve been shown to have more negative effects
Referrence-
1- Lecture by Prof Halawa Ahmed
2- C Frohn et al. The effects of HLA C on acute renal transplant rejection. Nephro Dial transplant. 2001 Feb;16(2):355-60.
Thanks, Dr Khan for your excellent answer.
I agree regarding the HLA Cw. I will start worrying if the DSA is more than 5000 or there is history of previous transplant.
What are the techniques used to detect DSA?
-Cell based methods and includes:
1- The complement dependent cytotoxicity (
This method detecting complement fixing antibodies.
this is old and not accurate
2- Flow cytometry:
A negative CDC result and a positive flow cytometry indicating that there is non complement fixing antibodies, non HLA antibodies or Low level DSAs.
-Solid phase antibody detection assays:
1-Enzyme-linked immunosorbent assay (ELISA):
2- Bead technology (Luminex):
It is a semi-quantitative test which is very sensitive to determine presence, strength and specificity of DSAs.
How you manage his transplantation?
it is considered high risk
induction with ATG followed by conventional triple immunosuppression Prednisolone,MMF and Tacrolimus.
Relevance of CW15 with MFI 1300
it has been shown that patients with high pre-transplant HLA-C DSAs have higher incidence of AMR in first year post-transplant , also associated with increased incidence of chronic AMR and poor graft survival. But here the level is low , only need monitored post transplant
What are the techniques used to detect DSA?
1-complement dependent cytotoxicity (CDC) cross match: is the oldest test in the HLA laboratory done by donor lymphocytes from blood or lymphoid tissue, incubating donor cells with recipient serum followed by rabbit complement and adding dyes to distinguish dead from living donor cells.
2-flow cross match involves the incubation of donor cells with recipient serum but, instead of complement, a fluorochrome-labelled anti-human IgG is added. This detector antibody will bind to antibodies which have been bound on the donor cell surface. In addition, fluorescent labelled antibodies specific to B and T lymphocytes are added to the donor cells then laser excitation identifies the lymphocytes and the presence of the detector antibody on the cell surface.
NB: The specificity of DSA cannot be easily determined using cross match assays because usually more than one HLA are expressed on donor cells.
3-Solid phase technology :
HLA source is manufactured beads coated with multiple HLA class I or II antigens (Phenotype beads or PRA beads) or a single HLA antigen [single antigen beads (SAB)]. HLA antibody testing using solid phase assays involves incubating the beads with the recipient serum and adding a fluorochrome-labelled anti-human IgG secondary antibody. The fluorescent signal can be detected using a flow cytometer, or more commonly, a Luminex analyser which is a semi-quantitative test which identifies the presence, the relative strength and the specificity of HLA antibodies.
How you manage his transplantation?
In this scenario the donor is living related with the same ABO group (compatabable )
a negative FCXM
111 mismatch and a low MFi not enough to cause positive crossmatch .as the cut value at which MFI is significant to cause ABR is variable but mostly MFI below 1500 is considered insignificant.
So no contraindication for transplantation.
There is 3 mismatch with 1 mismatch at DR so i will give Induction with ATG and maintain the patient on triple immunosuppression .
And i think we should do protocol biopsies at 1
,3,6,and 12 months post transplantation as there high incidence of recurrence in 60% of patients especially in living donors.
Two donor factors have emerged as possible risk factors for IgAN recurrence, donor source and donor IgA deposits.
Few studies found an increased rate of recurrence in patients with a living related donor compared with a deceased donor.but living donor is not contraindication for transplantation.
Two Australian registry studies have found that those with 1 or more HLA mismatches have a reduced rate of recurrence compared with those with zero mismatch kidneys,Similarly, ABO incompatible transplants have been found to have lower rates of recurrence, possibly due to differences in immunosuppression regimens.
One single-center Japanese study found that IgA deposits in the donor, presumably subclinical, increased the risk of IgA recurrence in the recipient.
What is the relevance of CW15 with MFI 1300?
high pre-transplant HLA-C DSA was associated with high incidence of AMR in the first year after transplantation.
Also increase risk of chronic AMR and poor graft survival.
So follow up of DSA post transplantation is recommended although MFI is low .
Reference :
Wyld, Melanie L. Chadban, Steven J. Recurrent IgA Nephropathy After Kidney Transplantation
.Transplantation: September 2016 – Volume 100 – Issue 9 – p 1827-1832.
Thanks, Shereen
I do not think that a protocol biopsy is warranted. This patient is low risk. I will start worrying if the DSA is more than 5000 or there is a history of a previous transplant.
– Complement-dependent cytotoxicity crossmatch
– Flow cytometry crossmatch
– Virtual cross match
Management plan of this transplant
=========================
This patient has a living-related donor, ABO compatible 1.1.1 mismatch, – ve FCXM with DSA + VE for Cw 15 at MFI 1300 so I will proceed with transplantation with induction by ATG and maintenance with triple IS ( CNI , MMF and steroid).
Regular monitoring of DSA post-transplantation.
HLA-C is a major determinant for NK cell activity
Many factors, both intrinsic and extrinsic to the HLA-C gene and HLA-C protein, explain its lower expression in comparison with HLA-A and -B. This lower expression can explain the apparent lower immunogenicity of HLA-C leading to a lower prevalence and strength of anti-HLA-C antibodies. Nevertheless, HLA-C antigens are truly immunogenic and preformed anti-HLA-C DSA are clinically relevant. Indeed, anti-HLA-C DSA is able to bind donor cells and activate the complement pathway both ex vivo and in vivo. In line with this, numerous clinical studies now show that preformed DSA directed at native HLA-C molecules induces poorer graft outcomes. However, with MFI 1300, the index is quite low so the impact is less but still has a negative impact on the graft
reference:
Visentin J, Couzi L, Taupin JL. Clinical relevance of donor-specific antibodies directed at HLA-C: A long road to acceptance. HLA. 2021 Jan;97(1):3-14. doi: 10.1111/tan.14106. Epub 2020 Oct 23. PMID: 33052032.
Thanks, Mike
These are the techniques of crossmatch. See other colleagues above
SAB is the technique of choice in detecting DSA.
1-CDC
flow cytometry & luminex
2-mismatch 3
living donor
DSA low MFI
induction ATG &maintenace with Tac,MMF,cortisol (as patient original kidney disease IGA and has high incidence for recurrence)
3-HLA C patient with DSA aganist HLA C specially with HLA B mismatch has high risk for AB mediated rejection specially in fist year
Thank you, Wael for your reply. It is always nice to see you contributing.
CDC is an old fashion and insensitive test for DSA.
YOU NEED TO EXPAND YOUR ANSWER AND ADD REFERENCES
Methods for HLA antibody screening include cytotoxic (cell-based) and solid phase antibody screening. (1)
A) Cell based (cytotoxic) method: It involves mixing recipient serum in a well containing 30-40 different ‘donor’ lymphocytes serum with complement and a dye leading to cell lysis (if antibodies are present in the recipient serum) which is observed under a phase contrast microscope. The degree of cell lysis is expressed as panel reactive antibody (PRA). Disadvantages with this method include lack of adequate representation of all the HLA types present in the population; presence of false postive values due to autoantibodies, IgM, and non-HLA antibodies. False negative results can be seen in conditions with low antibody titres. Prices documentation of unacceptable antigens cannot be done with this method.
B) Solid phase antibody screening: It involves using HLA molecules in place of lymphocytes.
1) Enzyme linked immunosorbent assay (ELISA): It is a 3 step procedure in which recipient serum is added to HLA glycoprotein labelled microtiter wells and after giving a wash, anti IgG with a passenger reporter molecule (alkaline phosphatase) is added followed by a wash. The third step involves addition of a substrate which undergoes a color change (due to dephosphorylation by the reporter molecule) if antibodies are present.
2) Microbead/ single-antigen bead technology: It is a very sensitive method in which recipient serum is added to beads labelled with fluorescein (reporter dye) and incorporated with HLA molecules. After giving a wash, Anti IgG lebelled with phycoerythron (detector antibody) is added and the result can be visualized using 2 laser beams, each detecting the reporter dye and specific bead. The results can be interpreted as either channel shift associated with the antibody binding (flowcytometry) or degree of fluorescence (mean fluorescence intensity, MFI) using Luminex method. It is useful in conditions with low level of DSA, non-HLA antibodies and non-complement binding antibodies, seen in a situation whereby CDC crossmatch comes out negative. The drawback with this method is that the bead kits available might not be representative of the HLA antigens in the community. They are specific, rapid, efficient, have increased range of identification (upto 11 HLA loci) and enable virtual crossmatch. But these methods are too sensitive, the antibodies detected might not have a clinical relevance. A false negative value can be seen due to high titre antibodies (prozone phenomenon), presence of IgM, IVIG use and due to epitope sharing (leading to reduction in MFI).
This is a live related, ABO compatible transplant with 111 mismatch and a negative FCXM. DSAw15) is present, but with low MFI (1300) DSA with MFI more than 3000 have been shown to be associated with increased antibody mediated rejection (AMR). (2)
Hence, this transplant can be done with Induction in form of ATG and triple drug immunosuppression (tacrolimus, MMF and steroids). Post-transplant DSA monitoring will be needed.
Another issue which also needs to be discussed with the patient is risk of recurrence, as IgA nephropathy has been shown to be associated with increased risk of recurrence in living related renal transplant. HLA B35 and DR4 are associated with increased risk of recurrence and decreased graft survival is seen with HLA B8 and DR3.(3,4) A better DR matching is also associated with reduced graft survival.(5) So the HLA typing of the patient is important.
Role of anti HLA-C antibodies in transplant has been evaluated and it has been shown that patients with high pre-transplant HLA-C DSAs have higher incidence of AMR in first year post-transplant. (6) They have also been shown to be associated with increased incidence of chronic AMR and poor graft survival. (7,8)
Since the level of DSA is less than 3000, the risk of AMR is low in this case. But post-transplant DSA monitoring will be important.
References:
1) Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and crossmatch: A comprehensive review. World J Transplant. 2017 Dec 24;7(6):339-348. doi: 10.5500/wjt.v7.i6.339. PMID: 29312863; PMCID: PMC5743871.
2) Malheiro J, Tafulo S, Dias L, Martins LS, Fonseca I, Beirão I, Castro-Henriques A, Cabrita A. Analysis of preformed donor-specific anti-HLA antibodies characteristics for prediction of antibody-mediated rejection in kidney transplantation. Transpl Immunol. 2015 Mar;32(2):66-71. doi: 10.1016/j.trim.2015.01.002. Epub 2015 Feb 7. PMID: 25661873.
3) Brensilver JM, Mallat S, Scholes J, McCabe R. Recurrent IgA nephropathy in living-related donor transplantation: recurrence or transmission of familial disease? Am J Kidney Dis. 1988 Aug;12(2):147-51. doi: 10.1016/s0272-6386(88)80010-6. PMID: 3041801.
4) Andresdottir MB, Haasnoot GW, Persijn GG, Claas FH. HLA-B8, DR3: a new risk factor for graft failure after renal transplantation in patients with underlying immunoglobulin A nephropathy. Clin Transplant. 2009 Sep-Oct;23(5):660-5. doi: 10.1111/j.1399-0012.2009.01059.x. Epub 2009 Aug 11. PMID: 19674013.
5) Bumgardner GL, Amend WC, Ascher NL, Vincenti FG. Single-center long-term results of renal transplantation for IgA nephropathy. Transplantation. 1998 Apr 27;65(8):1053-60. doi: 10.1097/00007890-199804270-00008. PMID: 9583865.
6) Aubert O, Bories MC, Suberbielle C, Snanoudj R, Anglicheau D, Rabant M, Martinez F, Scemla A, Legendre C, Sberro-Soussan R. Risk of antibody-mediated rejection in kidney transplant recipients with anti-HLA-C donor-specific antibodies. Am J Transplant. 2014 Jun;14(6):1439-45. doi: 10.1111/ajt.12709. Epub 2014 May 7. PMID: 24804568.
7) Visentin J, Bachelet T, Aubert O, Del Bello A, Martinez C, Jambon F, Guidicelli G, Ralazamahaleo M, Bouthemy C, Cargou M, Congy-Jolivet N, Nong T, Lee JH, Sberro-Soussan R, Couzi L, Kamar N, Legendre C, Merville P, Taupin JL. Reassessment of the clinical impact of preformed donor-specific anti-HLA-Cw antibodies in kidney transplantation. Am J Transplant. 2020 May;20(5):1365-1374. doi: 10.1111/ajt.15766. Epub 2020 Jan 18. PMID: 31883413.
8) Bachelet T, Martinez C, Del Bello A, Couzi L, Kejji S, Guidicelli G, Lepreux S, Visentin J, Congy-Jolivet N, Rostaing L, Taupin JL, Kamar N, Merville P. Deleterious Impact of Donor-Specific Anti-HLA Antibodies Toward HLA-Cw and HLA-DP in Kidney Transplantation. Transplantation. 2016 Jan;100(1):159-66. doi: 10.1097/TP.0000000000000821. PMID: 26262501.
good. Would you recommend protocol biopsies post transplant?
As the MFI level is low, I would not be performing a protocol biopsy in this patient. But DSA monitoring would be needed.
I would recommend DSA minitoring rather than protocol biopsy
Dear All
How often we use CDC technique in detecting DSA?
What is the sensitivity and specificity of CDC in quantifying DSA qualitatively and quantitatively?
CDC technique in DSA detection is now less used than before and recent solid phase assay replaced it(e.g. SBA) which is more sensitive than CDC.
Using CDC with an AHG keep its highly specificity than SPI.
So its old but still gold (1).
1-Complement‐Dependent Cytotoxicity (CDC) to Detect Anti‐HLA Antibodies: Old but GoldPatrícia Keiko Saito,et al
In our center we are still often using it.
Although adding AHG can increase its sensitivity as compared to the stansard CDC, BUT IT IS STILL RELATIVELY INSENSITIVE AS COMPARED TO NEWER TECHNIQUES LIKE FCXM & SOLID PHASE ASSAY.CDC THOUGH LESS SENSITIVE ,IT HAS HIGH SPECIFCITY.
Thanks, Dr Mohamed
Try to push for employing SAB in detecting DSA in your centre.
Thank you for your reflection Dr Mohamed.
CDC is no more in use in UK, but srill used in other countries. Its not specific and not sensitive, as its not detecting non complement fixing DSA and as we know significant proportion of DSA is non complement fixing, therefore it will net be detected . Furthermore its not detecting the lower levels of DSA. So it’s nonsensitive..
Dear Dr Wael
Please reflect on this statement of yours please” CDC is not detecting non-complement fixing DSA”
Dear Dr. Ahmad, what I meant is that CDC will detect only Complement fixing DSA while the LSAB will detect complement fixing and noncomplement fixing DSAs.
Thank you
CDC is an old method, the gold standard method now is the luminex SAB which is more sensitive and specific.
CDC cross match is the oldest test in the HLA laboratory and involves extracting donor lymphocytes from blood or lymphoid tissue, incubating donor cells with recipient serum followed by rabbit complement and adding dyes to distinguish dead from living donor cells. This process detects the presence of antibody-antigen interaction on cell surface which activates complement and cause cell death.
Some centers still use it but in most centres Luminex SAB is used.
In most cases a positive cross match, either with CDC or flow cross match, indicates DSA binding to the donor cells. This is not always true as auto antibodies or unknown non-HLA factors might cause a false positive cross match.
The specificity of DSA cannot be easily determined using cross match assays because usually more than one HLA are expressed on donor cells
But Using CDC with an AHG increases its specificity .
It is also not sensitive as it cant detect non complement fixing DSA .
Jennifer McCaughan, Qingyong Xu, and Kathryn Tinckam.Detecting donor-specific antibodies: the importance of sorting the wheat from the chaff
CDC techniques is old method not sensitive or specific detect only complement fixing antibodies
But Solid phase assay (SAB) by luminex is gold standered detect both complement fixing and non fixing DSA.
my colleague ask me in presence of Luminex and flow cytometry which is highly sensitive than CDC why we still use CDC ?
On other hand negative luminex and flow cytometry expect to be positive by CDC ?
My answer : this prospect may be true for negative result but those sensitize with very high MFI and flow cytometry we have two approach,
*if positive CDC crossmatch so patient contraindication to transplantation .(relative or absolute according to type B or T lymphocyte ) .
*if negative CDC patient may go ahead for transplant with desensitization regimen(plasma exchange or immune absorption technique ).
The technigues used to detect DSAs are complement dependent cytotoxicity CDC which is no more valid in most of transplant centers. And then the serology technigue including Eliza which is less accurate than the Solid phase assay. Flow cytometry cross match is detecting all anti_HLA antibodies regardless to being complement fixing or not. Luminex which is single antigen bead is highly specific and sensitive technique. We can proceed with transplantation as the donor recepient is 111 mismatch with MFI of 1300 for class 1HLA antigen. Protocol will be ATG induction and triple Tacrolimus based maintenance protocol. CW15 antibody of 1300 is not significant as our cut off for class 1 antibody is 2000 MFI, however I would advise to follow up for DSAs regularly post transplantation.
Thanks, Dr Wael
I appreciate your experience. This patient is low risk. I will start worrying if the DSA is more than 5000 or there is a history of a previous transplant.
1- Techniques for DSA detection
Serological techniques : complement dependant cytotoxicity cross match
Solid phase technology including flowcytomtery and luminex technology as single antigen assay.
2- Management plan
This patient has a living related donor , ABO compitable 1.1.1 mismatch , – ve FCXM with DSA + VE for Cw 15 at MFI 1300 proceed with transplantation with induction by ATG and maintenance with triple IS ( CNI , MMF and steroid).
Regular monitoring of DSA posttransplantation.
3- Presence of preformed cw Ab
Is associated with increased immunological risk and risk of AMR and adverse graft outcome.
Dear all
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Is there any difference in the risk of ABMR with Anti Native and Anti- denatured Anti -HLA C antibodies?
Excellent response, thank you, Dr Mohamed Mohamed
Thanks a lot Prof Ala
Dear Dr Mohamed
Can you prepare a talk on the relevance of HLA C in transplantation to all of us?
We can arrange a zoom meeting and invite all colleagues on the course.
The main feature of HLA C antigen is its lower expression in comparison to HLA A and HLA B antigens. Which is due to many factors related to the HLA C gene and HLA C protein itself. This lower expression can explain can explain the apparent lower immunogenicity of HLA C antigens leading to a lower prevalence and strength of anti HLA C antibodies. However, anti HLA C DSA are able to bind donor cells and to activate the complement cascade potently. Therefore, preformed anti HLA C antibodies is associated with poor allograft outcome shown by numerous clinical studies.
I think the risk of AMR is more with anti denatured HLA C antibody as theoretically it might be be mounted against a wider array of epitops than for anti normal HLA C antibidies. Just an opinion.
Many factors, both intrinsic and extrinsic to the HLA-C gene and HLA-C protein, explain its lower expression in comparison with HLA-A and -B. This lower expression can explain the apparent lower immunogenicity of HLA-C leading to a lower prevalence and strength of anti-HLA-C antibodies. Nevertheless, HLA-C antigens are truly immunogenic and preformed anti-HLA-C DSA are clinically relevant.
Indeed, anti-HLA-C DSA are able to bind donor cells and to activate the complement pathway both ex vivo and in vivo. In line with this, numerous clinical studies now show that preformed DSA directed at native HLA-C molecules induce poorer graft outcomes.
Thanks, Dr Zahid
But you did not tell us what are you going to do. What is your plan for this patient?
References:
Thank you
although anti HLACW15 less immunogenic or harm than anti HLA A,B,DR but recent study prove that it has deleterious effect on graft and this adverse impact more with anti native than with anti denatured so optimum way to do SAB to differentiate between two.
What are the techniques used to detect DSA?
Cytotoxic method, using a reactive panel of antibodies with donor lymphocytes and patient serum. The most specific methods are solid phase, mainly ELISA, flow cytometry, and single antigen beads. These can calculate a PRA (cPRA) with higher specificity of detecting the Crossmatch and is based on unacceptable HLA antigens.
How you manage his transplantation?
IgA nephropathy may be the reason for DSA positivity, but the patient already has 50% HLA mismatch.
I would do lymphoablative induction (ATG or alemtuzumab) associated with a maintenance treatment with a triple regimen (Tacrolimus, MMF plus prednisone)
What is the relevance of CW15 with MFI 1300?
HLA-C has an uncertain behavior in the literature, but the values in question are low, so we can consider the programmed dosage of DSA in the post-transplantation associated with on-demand graft biopsies.
Agree
Antibodies against HLA can be detected by several methods:
(b) microbead/ single Ag beads
This recipient had 50% mismatch with negative flow cytometry, but low DSA against HLA-Cw Ag, so he need induction with T cell depleting agent + IVIG & maintained on CNI+ MMF+ steroid, with follow up of DSA.
Traditionally, HLA-Cw considered as low immunogenic when compared to Class I & II HLA ( HLA-A, -B, -DR), & this may be due to low expression of HLA-Cw Ags on cell surface. But recent studies proved that the DSA against HLA-Cw had the same impact on graft survival as other DSA against class I &II Ags. Also it was found that presensitized recipient ( HLA-Cw DSA) that use for induction ATG in addition to IVIG had low risk of rejection.
References:
Santos S., Malherio J., Tafulo S., et al. Impact of preformed donor-specific antibodies against HLA class I on kidney graft outcome: Comparative analysis of exclusive anti Cw vs anti-A and /or -B antibodies. World J Transplant , 2016;6(4):689-696.
Our case with a diagnosis of IgA nephropathy, on HD
he has an offer from LRD {son}, with 111 mismatches, compatible ABO, Negative FCXM, DSA positive with low MFI 1300
Q1: What are the techniques used to detect DSA?
1- Cytotoxic (cell-based) method CDC:
a complement-dependent assay that requires high titer antibodies for a complement to be activated so false-negative results can occur due to low titer Abs. it is not specific for HLA as it can be falsely positive due to non-HLA Abs, auto-antibodies and IgM Abs.
2- Solid-phase Antibody screen:
a- ELISA or
b- Microbead platform(Flow cytomere) /single antigen beads SAB (Luminex-SAB)
are sensitive and specific for HLA antibody
can detect low titer DSA or complement non-fixing that can be clinically insignificant, to overcome this; C1q assay can be done which will identify only complement-fixing antibodies that are clinically significant. (expensive)
How you manage his transplant?
I will consider him high risk because he has HLA-DR mismatch and has a DSA positive.
induction with ATG, methylprednisolone
maintenance with Tac+ prednisolone+MMF
What is the relevance of CW15 with MFI 1300?
there is no consensus about the role of HLA-C in the outcome of transplantation, our patient has a low titer that will make him candadete for desensitization before transplant, but I will follow him with DSA monitoring and biopsy according to the DSA and clinical parameters.
Thanks, Jamila
Agree
What are the techniques used to detect DSA?
Complement-dependent cytotoxicity assay.
Enzymelinkedimmunoabsorption.
Flow cytometry: multiplexed particle-based (Luminex).
Single antigen beads .
Anti-HLA-Cw before are considered less immunogenic and are not considered in
many organ allocation systems or immunologic risk stratification algorithms. However,
data from literature confirms that their presence is as deleterious as DSA anti-HLA
A/B/DR/DQ. Thus we should take HLA-C typing and respective antibody identification.,
in order to access risk stratification and establish the need for correct induction or
desensitization therapies.(1)
Type of Anti-HLA cw:
Anti-denatured HLA-Cw antibodies are highly prevalent, whereas anti-native HLA-Cw
antibodies seem to lead to random flow cytometry crossmatch results.
Anti-native but not anti-denatured HLA-Cw antibodies are deleterious, Those with anti-
native HLA antibodies experienced more acute and chronic antibody-mediated rejections
, and displayed a lower graft survival ,. Patients with anti-native HLA-Cw antibodies
more frequently had previous sensitizing events which underscores the need for
reagents with diminished expression of denatured HLA.(2)
Either ATG or Alemtuzumab can be used plus standard IS.
Its low level transplant can be done without desensitization.
References:
1- Kidney transplantation in patients with preformed and exclusively anti-HLA-
Cw donor specific antibody Sofia Santos1, Ana Castro1, Andreia Campos1, Sofia
Pedroso1, Leonídio . Port J Nephrol Hypert. 2017; 31(1): 50-53 • Advance Access
blication 8 March 2017.
2-Jonathan Visentin,Thomas Bachelet,Olivier Aubert,Arnaud Del Bello,Charlie Martinez
et al. Reassessment of the clinical impact of preformed donor-specific anti-HLA-Cw
antibodies in kidney transplantation: 28 December 2019.https://doi.org/10.1111/ajt.15766
Citations: 10.
Good, thank you, Dr.Mohamed
SAB is more sensitive Dr Mohamed
What are the techniques used to detect DSA? HLA typing by Serology any Molecular study (PCR), molecular typing help to identify unique HLA alleles.
Method of desensitization: by use CDC
Punal reactive Antibody
Solid phase antibody by ELSA & Single antibody bead used to detect antibody by flow cytometry or by single antigen bead luminex platform ( this method detect specific HLA Ab-Ag) & specific for DSA. C-PRA is best to identify incompatible donor and called positive cross match. Microbead assay ( flow PRA) more sensitive to detect DSA & SAB help to identify complement & non complement binding.
How to Manage his transplant? this patient is a father of son which are ABO compatible and flow cytometry negative but DSA positive for Cw15 with MFI 1300 / mismatch 1.1.1
he’s had intermediate immunological risk on long term of graft. So need aggressive immunosuppressive agents pre & post transplant by Anti thymocyte globulin & may need plasma exchange immediately post transplant 3 to 5 sessions and Maintenance treatments by triple agent ( tacrolimus / cellcept/ steroid
What is the relevance of Cw15 with MFI 1300.
HLA- C is nonshared allotype but it can lead to allograft rejection. so desensitization protocol needed to identify rare alleles. also the mechanism of HLA c antigen on outcome of survival allograft still unknown. the most important HLA A, B, DR in kidney transplant especially HLA DR matching has great effects on graft than HLA A, B. but presence of HLA c still has risk of immunological effect on graft for long term survival of graft so adherence to immunosuppressive agents mandatory to avoid graft rejection.
References:
1. A. Woolfrey, et al. HLA-C antigen mismatch is associated with worse outcome: Biol Blood Marrow Transplant. 2011.Copyright © 2011 American Society for Blood and Marrow Transplantation.
2. C Frohn et al. The effect of HLA-C matching on acute renal transplant rejection: Nephrol Dial Transplant. 2001 Feb;16(2):355-60
Cytotoxic cell based antibody screening: Has several limitations, recipient serum is mixed with panel of different donor lymphocytes in individual wells with complement and dye, serum antibodies bind to cell surface leading to cell death, degree of cytotoxicity is determined as percentage PRA
Solid phase antibody screening: using recombinant HLA molecules through 2 methods,
ELISA: purified HLA molecules are applied to ELISA platforms and bind HLA antibodies in recipient serum
Single antigen beads: beads are coated with class I or II HLA antigens, microbead conjugated with fluorescent dye detect presence of HLA antibodies, fluorescence detected through flow cytometer (Flow PRA) or SAB Luminex, both are more sensitive than ELISA
It is considered a standard risk transplant as
living donor, first transplant
FCXM is negative, although have preformed anti-HLA-Cw DSA positive but with low MFI.
HLA typing with 111 mismatch including HLA-DR mismatch which has impact on graft survival.
Induction therapy with Basiliximab (used in induction in low to moderate risk to prevent acute rejection with less side effects)
maintenance therapy:
CNI: Tacrolimus with keeping trough level 7-10 in first three months and 5-7ng/mL in next months
mycophenolic acid
Corticosteroids: methylprednisolone pulse then gradual tapering to reach low oral dose daily.
Steroid withdrawal should be avoided as it is associated with increased rate of IgA nephropathy recurrence post transplant.
the association between antithymocyte globulin induction and IgA nephropathy recurrence post transplant is controversial.
Clinical relevance of preformed anti-HLA-Cw antibodies is recently recognized, they were initially considered non pathogenic but with improvement of detection techniques (SAB), their impact was better investigated.
A retrospective case controlled multi-center study assessed clinical impact of isolated anti-HLA-Cw antibodies and showed that they were associated with similar incidence of AMR and same graft survival as anti-HLA-A,B,DR and DQ antibodies
More than 10% of positive HLA-Cw beads could correspond to anti-denatured-HLA antibodies and are suspected to be clinically irrelevant.
Preformed anti-native HLA-Cw antibodies are associated with higher incidence of AMR and had lower graft survival.
Althaf MM, El Kossi M, Jin JK, Sharma A, Halawa AM. Human leukocyte antigen typing and crossmatch: A comprehensive review. World J Transplant 2017; 7(6): 339-348
Wyld ML, Chadban SJ. Recurrent IgA nephropathy after kidney transplantation. Transplantation. 2016 Sep 1;100(9):1827-32.
Leeaphorn, N., Garg, N., Khankin, E.V., Cardarelli, F. and Pavlakis, M., 2018. Recurrence of IgA nephropathy after kidney transplantation in steroid continuation versus early steroid‐withdrawal regimens: a retrospective analysis of the UNOS/OPTN database. Transplant International, 31(2), pp.175-186.
Bachelet T, Martinez C, Del Bello A, et al. Deleterious impact of donor-specific anti-HLA antibodies toward HLA-Cw and HLA-DP in kidney transplantation. Transplantation. 2016;100(1):159-166.
Visentin J, Bachelet T, Aubert O, Del Bello A, Martinez C, Jambon F, Guidicelli G, Ralazamahaleo M, Bouthemy C, Cargou M, Congy‐Jolivet N. Reassessment of the clinical impact of preformed donor‐specific anti‐HLA‐Cw antibodies in kidney transplantation. American Journal of Transplantation. 2020 May;20(5):1365-74.
McCaughan J, Xu Q, Tinckam K. Detecting donor-specific antibodies: the importance of sorting the wheat from the chaff. Hepatobiliary Surgery and Nutrition. 2019 Feb;8(1):37.
Thanks Heba
What are the techniques used to detect DSA?
Cytotoxic (cell-based) antibody screening
False-positive results can be produced due to non-HLA antibodies, autoantibodies, and nonspecific IgM antibodies. False-negative results are possible because it requires high antibody titers to activate complement.
Solid-phase antibody screening
Enzyme-linked immunosorbent assay platform (ELISA):
Purified HLA molecules are applied to ELISA platforms and will bind individually to HLA antibodies.
Microbead platform/single-antigen beads( SAB): SAB assays are rapid with
results available in 3-4 h.
-Solid-phase antibody screening can detect both complement and non-complement binding antibodies.
-It sensitive can detect antibody that is below the threshold associated with a positive crossmatch.
How you manage his transplantation?
Both donor and recipient are ABO compatible with negative FCXM but low titer DSA. They have a 1.1.1 mismatch.
This recipient has a high risk for acute rejection. He needs induction with ATG and follows with maintenance immunosuppression TAC, MMF, and steroid.
-close follow-up for DSA.
What is the relevance of CW15 with MFI 1300?
HLA-C mismatch is significantly correlated with acute transplant rejection with an additional mismatch on the B locus. One retrospective study shows that two years after transplantation, the biopsy-proven acute rejection-free survival was worse in the Cw/DP DSA than in the no DSA group. Accordingly, graft survival was lower in the Cw/DP than in the no DSA group .
Christoph Frohn ,Lutz Fricke ,Jan-Christoph Schlage-Puchta .The effect of HLA-C matching on acute renal transplant rejection. March 2001Nephrology Dialysis Transplantation 16(2):355-60.
Thomas Bachelet 1, Charlie Martinez, Arnaud Del Bello, Lionel Couzi, et al . Deleterious Impact of Donor-Specific Anti-HLA Antibodies Toward HLA-Cw and HLA-DP in Kidney Transplantation . Transplantation . 2016 Jan;100(1):159-66.
Thanks Reem
You can see that there is no consensus regarding HLA C DSA
What are the techniques used to detect DSA?
1- complement dependent cytotoxicity(CDC) : an old test occasional false +ve and false -ve results.
2- flow cytometry : for single antigen assay, more sensitive.
3- solid phase assay : as Luminex. it is highly sensitive
How you manage his transplantation?
1- induction therapy with ATG and methyl prednisolone.
2- maintenance therapy with Cyclosporine, MMF, Prednisolone. …. Cyclosporine and steroids in the 1 st year, are more helpful for prevention of IgA recurrence in kidney graft.
3- close monitoring of DSA in the post-transplantation period.
4- protocol biospy should be considered.
What is the relevance of CW15 with MFI 1300?
1- DSA for HLA-C has been known to be of low significance in solid organ transplantation. however, it was later found to have a serious role in highly sensitized recipients as it can be involved in pathogenesis of chronic AMR.
2- the titre is less than 2000 and the cross match test is -ve. therefore, its role in this scenario is not important
Excellent
techniques used to detect DSA
-CDC (complement dependent cytotoxicity)
false negative in low titer
canot differentiate between HLA from non HLA
Auto Ab and IgM give false positive
solid phase assay :
– ELISA
differennce between kit and other( no fixed standardization)
-flow cytometry
detect Ab to T and B lymphocyte
highly sensitive
-Luminex (single antigen bead)
commonly used highly sensitive
may missed non HLA Ab
*how to manage transplantation ?
patient sensitized since he has DSA
although risk not that of anti A,B,DR but not standard immunological risk
AII what we need INTENSI IMMUNOSUPRESSIVE REGIMEN
include ATG induction ,TAC ,MMF, STEROID with close monitoring DSA post transplantation
-Question here we need full desensitization protocol (including plasma exchange )or immediate transplantation ?
I think immediate transplantation since low titer (slight increase 1300MFI) in sensitized with anti HLA class 1 we can proceed for transplantation if titer below 1000 MFI .
*Relevance of anti CW15?
previous prospect consider antiCW15 non immunogenic but with advance in Ab screening technique anti CW15 can cause rejection and carry adverse impact on graft survival .
to make full assessment of DSA we need to know in addition to titer
sub class ,c1q ,type and titer .
ing M, Marfo K, Masiakos P, Aljanabi A, Lindower J, Glicklich D, et al. Pretransplant
anti-HLA-Cw and anti-HLA-DP antibodies in sensitized patients. Hum Immunol.
2012;73(9):879-83. 3.
Thorsby E, Sandberg L, Lindholm A, Kissmeyer-Nielsen F. The HL-A system: evidence of a third sub-locus. Scand J Haematol. 1970;7(3):195-200. 4.
Gilbert M, Paul S, Perrat G, Giannoli C, Pouteil Noble C, Morelon E, et al. Impact of
pretransplant human leukocyte antigen-C and -DP antibodies on kidney graft outcom
The techniques that are used to detect DSAs include:
-Solid phase assays: Luminex-based studies such as the single antigen (SA) assay and the Luminex cross match assay are the most sensitive techniques.
-Complement-dependent cytotoxicity (CDC) cross match is an old test used to identify DSAs.
-Single antigen assays in the flow cytometry cross match .
Management plan:
Induction by ATG 3-5 doses, pulse steroids then maintenance on Tac, MMF and prednisolone with follow up of DSA level after transplantation and may be protocol biopsy.
Relevance of CW15 with MFI 1300:
HLA-C mismatch was related to reduced graft survival in sensitized recipients ,Due to the low MFI and more importantly the fact that the FCX is -ve , so definitely no desensitization protocol should be given .
References:
1- Sofia Santos1, Ana Castro1, Andreia Campos1, Sofia Pedroso1, Leonídio Dias1, Castro Henriques1. Kidney transplantation in patients with preformed and exclusively anti-HLA-Cw donor specific antibody. Port J Nephrol Hypert 2017; 31(1): 50-53 • Advance Access publication 8 March 2017.
2-Thomas B; Charlie M; Arnaud DB; Lionel C, et al.Deleterious Impact of Donor-Specific Anti-HLA Antibodies Toward HLA-Cw and HLA-DP in Kidney Transplantation
Thanks Mohamed
*What are the techniques used to detect DSA?
-Serological
Less accurate ,less standardized and oldest
methods
Complement depedant cytotoxicity
-Solid phase assay
More sensitive than serological method
ELISA
There is variation between the kits
Flowcytometry crosmath
Luminex single antigen beeds commonly used
*How you manage this transplantation?
-ABO compatible
-HLA 1-1-1 (A,B,DR)
-FCXM negative
-DSA positive in anti CW 15 with MFI 1300
which is less than cut off value for class1 (2000 MFI) ,, but still there is possibility of high immunological risk
I manage this transplantation with induction therapy ATG ,Methyle prednisolone
maintenance Tacrolimus , MMF ,Prednisolone ,with reqular fellow up (DSA) and biopsy for the risk of AMR
*What is the relevance of CW15 with MFI 1300 ?
There is no clrea role regarding anti HLA- C antibodies but many studies showed it has higher risk for AMR
(In conclusion, patients with pretransplant HLA-C DSA appear to be at higher risk for AMR development. Pretransplant risk stratification of sensitized patients may be accomplished by testing for donor-specific HLA-C antibodies. Screening is therefore necessary, and modulation of immunosuppression should be required in cases of positivity. Future prospective studies in larger cohorts and with longer follow-up periods are needed to further validate these observations.
https://onlinelibrary.wiley.com/journal/16006143
Risk of Antibody-Mediated Rejection in Kidney Transplant Recipients With Anti-HLA-C Donor-Specific Antibodies
O. Aubert,M.-C. Bories,C. Suberbielle,R. Snanoudj,D. Anglicheau,M. Rabant,F. Martinez,A. Scemla,C. Legendre,R. Sberro-Soussan
First published: 07 May 2014
https://doi.org/10.1111/ajt.12709
Citations: 33
Other studies showed the importance of HLA -C matching to prevent acute rejection episode.
Conclusion. HLA‐C matching of all kidney donors and recipients seems to be an option to reduce the probability of acute rejection episodes.
The effect of HLA‐C matching on acute renal transplant rejection
Christoph Frohn, Lutz Fricke, Jan‐Christoph Puchta, Holger Kirchner
Nephrology Dialysis Transplantation, Volume 16, Issue 2, February 2001, Pages 355–360, https://doi.org/10.1093/ndt/16.2.355
Published: 01 February 2001
Thanks, Elaf for your effort, well done. A good start
Thanks prof Ahmed
Techniques used to detect DSA include
the complement-dependent cytotoxicity (CDC) crossmatch, which is the oldest test available in the HLA laboratory and is used to identify DSA.
Single antigen assays in the flow crossmatch and solid phase
Because of their excellent sensitivity and specificity, Luminex-based diagnostics, such as the single antigen (SA) assay and the Luminex crossmatch (Xm-DSA) assay, are the most often used techniques for detecting anti-HLA antibodies.
With MFI 1300, this is a son-to-father transplant. The donor and recipient are ABO compatible, with a 1.1.1 mismatch at the ABO donor-recipient ratio. The flow cytometry was negative for XM but positive for DSA to CW15 (which is low). As a result, we can go on with transplanting (ATG induction and maintenance on Tacrolimus mycophenolate and prednisolone and Follow up of DSA level post-transplantation).
What is the relevance of CW15 with MFI 1300?
The impact of HLA-C mismatch on transplant survival in pre-sensitized kidney recipients was investigated in a retrospective research study. It was discovered that having an HLA-C mismatch was related to considerably reduced graft survival in pre-sensitized recipients, but not in non-pre-sensitized recipients.
The risk of rejection is still present, so close monitoring of the DSA post-transplantation is recommended.
Thomas B; Charlie M; Arnaud DB; Lionel C, et al.Deleterious Impact of Donor-Specific Anti-HLA Antibodies Toward HLA-Cw and HLA-DP in Kidney Transplantation
Excellent
2. You have seen a 53-year-old CKD patient on HD due to IgA nephropathy who is fit for transplantation (all work up is acceptable). His son, 111 mismatch ABO compatible is willing to donate a kidney. FCXM is negative but DSA was identified (CW15 with MFI 1300)
What are the techniques used to detect DSA?
1. Complement based cytotoxic assays
and ELISA: older and less sensitive techniques
2. Luminex-based tests, such as the single
antigen (SA) assay and the Luminex crossmatch (Xm-DSA) assay: are the most
commonly used due to their high sensitivity and specificity.
3. Next-generation
targeted (NGT) sequencing assay to detect
donor-derived cell-free DNA after transplantation .
How you manage his transplantation?
I will manage his transplantation as
moderately high risk and proceed with induction using ATG followed with
conventional triple immuno-suppression (TAC,MMF& Steroids)
What is the relevance of CW15 with MFI 1300?
It remins unclear whether anti–HLA-Cw DSA really
exerts a pathogenic role and impact graft outcomes in kidney transplantation.
Only few studies focused on the outcome of patients transplanted with
preformed anti–HLA-Cw DSA. In a series of 20 patients transplanted with
preformed anti–HLA-Cw and/or anti–HLA-DP antibodies associated to other DSA,
acute rejection and immunologic graft loss were more frequent than in the 176
patients transplanted only with anti–HLA-A/B/DR/DQ antibodies. Bachelet T, Martinez C, Del Bello A,
Couzi L, Kejji S, Guidicelli G, Lepreux S, Visentin J, Congy-Jolivet N,
Rostaing L, Taupin JL, Kamar N, Merville P. Deleterious Impact of
Donor-Specific Anti-HLA Antibodies Toward HLA-Cw and HLA-DP in Kidney Transplantation.
Transplantation. 2016 Jan;100(1):159-66. doi: 10.1097/TP.0000000000000821.
PMID: 26262501.
Excellent Dr Mohamed
1-Method for DSA identification
1-T he Complement Dependent Cytotoxicity Crossmatch (CDC-XM)
The technique uses donor lymphocytes and recipient serum, which are incubated before addition of complement. It detects all complement fixing IgG, IgM antibodies of HLA and non-HLA origins as well as autoantibodies.
Importance of a T/B cell positive CDC-XM: The CDC-XM is performed separately on T and B cell lines. T lymphocytes carry only HLA class-I antigens. DSA against such HLA-I antigens are associated with significant risk of HAR and AMR .
Therefore, the T cell crossmatch is a crucial component in the CDC-XM. B cells carry both HLA class-I and class-II antigens. T herefore, a positive B cell crossmatch means DSA against either HLA class-I, II or both. A B-cell positive, T-cell negative crossmatch, signifies either DSA against HLA class-II only or low titre DSA against HLA class-I undetectable by T cells.
2-T he Flow-Cytometry Crossmatch (FCXM)
FCXM also depends on donor lymphocytes being incubated with recipient serum. However, instead of adding complement factors, a fluorescence-coated second antibody is added that acts against the IgG-DSA. This anti-IgG antibody binds to the donor Ag-DSA complex and allows detection through a flow-cytometer. The reading is semi-quantitative.
3-Solid Phase Immunoassay (SPI)
ELISA) ] or synthetic beads; (Luminex) ]. SPI is specific for HLA antibodies and thereby eliminates the false positives in CDC-XM and FCXM caused by non-HLA antibodies and autoantibodies. ELISA test is more sensitive than CDC-XM while the Luminex is more sensitive than both the CDC-XM and FCXM.
T he Luminex-SPI
This consists of a series of polystyrene microsphere beads to which target HLA antigens are attached after purification. The relevant beads are labelled with differing ratios of fluorescent dyes giving them a unique fluorescent signal. Test sera is added where any DSA present in the sera would bind to appropriate HLA molecules on beads. The resulting antigen-antibody binding can be detected via laser based fluorescent imaging quantified as Mean Fluorescent Intensity (MFI). The assay can be taken one step further with the Single Antigen Bead (SAB) test, where the relevant beads are all coated with a single cloned antigen. The SAB test is the most precise and specific in detecting DSA against a specific antigen.
2-the patient have more than one HLA mismatch so considered high risk
Induction with ATG or Alemtuzumab
Maintenance with TAG,MMF,Steroid
Negative cross match so No need for desensitization
3-case report by Sofia Santos1, Ana Castro1,etal show that
anti-HLA-Cw have deleterious effect as DSA anti-HLA A/B/DR/DQ.
Thus we should take HLA-C typing and respective antibody identification into account in sensitized patients, in order to access risk stratification and establish the need for correct induction or desensitization therapies.
Reference
1- Nalaka Gunawansa1,2, Roshni Rathore2,3, Ajay Sharma2,4 and Ahmed Halawa2,5*. Crossmatch Strategies in Renal Transplantation: A Practical Guide for the Practicing Clinician. J Transplant Surg 2017, 1(1):8-15
2- Sofia Santos1, Ana Castro1, Andreia Campos1, Sofia Pedroso1, Leonídio Dias1, Castro Henriques1. Kidney transplantation in patients with preformed and exclusively anti-HLA-Cw donor specific antibody. Port J Nephrol Hypert 2017; 31(1): 50-53 • Advance Access publication 8 March 2017.
Thanks, Dalia for your excellent reply
I’m still confused regarding the role of HLA C. It May be important in retransplantation.