II. Consensus Guidelines on the Testing and Clinical Management Issues Associated with HLA and Non-HLA Antibodies in Transplantation

• Please give a summary of this article

           
Introduction
 
Solid-phase immunoassay (SPI) detects & characterize HLA antibodies more sensitively than CDC assays.
 
Luminex assay (using SPI technology), can detect antibodies not detectable by CDC; the significance of these antibodies in practice is not well understood & its results needs careful consideration.
 
The detection of these antibodies, however has made changes in the clinical management of sensitized patients.
 
SPI testing also raised some technical issues that need to be resolved.   
 
Here is the consensus report & recommendation made by The Transplantation Society group of laboratory & clinical experts.
 
They used of best available evidence & expert opinion in doing this task.         
 
Consensus Recommendations
 
Technical Group
 
1) Antibody identification:
a. At least 1 SPI should be used for HLA class I & II-specific antibodies.
A SAB should be done at least once pre-transplantation in HLA-immunized patients.(esp. for antibodies directed at Cw, DQA, DPA, & DPB, which are not adequately defined by other techniques).
 
b. Use both SPI & cell-based assays to assess antibody status to the intended donor.
 
c. Laboratories must correlate the level of antibody detected by SPI with cell-based assays to establish the likelihood of a positive XM.
 
2) Standards for cell-based assays (CDC)
 
a. CDC assays for antibody identification & XM should be done using target cells that permit identification of antibodies to both HLA class I &II antigens.
 
b. Nonspecific reactivity must be recognized.
 
c. Consider modifications to increase sensitivity & specificity e.g.serum modification steps to remove or inactivate IgM & C1.
 
3) Standards for flow cytometry cell-based assays (flow cytometry)
a. Differentiation of T & B cells should be performed by a three-color fluorescence technique.b. Consider modifications such as Pronase use to increase sensitivity & specificity.

4) Standards of practice
 
a. The lab.must have documented expertise in antibody assessment & interpretation.
 
b. Lab. must establish its own threshold for antibody specificity assignment & clinical interpretation.
 
c. Each center should define changes in MFI values between sera from the same patient that are clinically meaningful.
 
d. The patient history must be considered for the interpretation of antibody screening & interpretation of test results; e.g. history of parity & previous graft HLA mismatches.
 
e. In determining antibody specificity, the lab.should consider the possibility of antibodies to epitopes on any polymorphic chains (including DQA & DPA) as well as epitopes resulting from combinations of different œ & ᵝ chains.
 
f. HLA typing of donor & recipient must be performed at a level required for accurate antibody interpretation.
 
g. Store donor material in the form of frozen cells & DNA for post-transplantation DSA investigations.
 
5) Interfering factors in interpretation of SPI
 
a. Consideration must be given to these variables when performing & assessing HLA antibody results:
 
  – antigen density on beads & condition (i.e., denatured Ag);
   – reactivity of control sera & control beads;
  – reduction of test interference (i.e., EDTA, DTT, & hypotonic dialysis);&
  – when saturation of target antigens may have occurred, sera should be
  tested under conditions where meaningful changes in antibody levels can
  be detected (e.g., serum dilutions).
 
6) Assay standardization
 
a. Labs should follow standardized operating procedures & policies that minimize test variability including, wherever possible, robotic processing, temperature control, consistency in washing procedures, & instrument calibration.
 
b. Quality-control procedures must be introduced to monitor inter-assay & intra-assay variability.
c. Each laboratory must participate in relevant external proficiency testing programs as required by local, regional, & national regulations.
 
7) Reporting of results
 
a. Reporting  format should include:
 
1. Sample & assay dates
2. Assay name
3. Calculated RF/cPRA/virtual PRA indicates the frequency of donors with unacceptable HLA antigen mismatches.
4. Specificity assignment & assessment of antibody level.
5. Comments on presence/absence of DSA if a specific donor is being assessed
6. Immunoglobulin class & isotype if available
7. Assay or serum modification employed.
 
Pretransplantation Group
 
8) Risk  stratification categories should be developed based on antibody identification & XM results.
 
9) Prior sensitizing events should be considered in interpreting antibody testing results.
 
10) DSA detected by CDC antibody screening & XM in the most recent serum collected must be avoided because they are associated with a high risk for AMR & graft loss.
 
11) To minimize risk of sensitization & antibody-mediated allograft damage, blood products transfusion should be avoided if possible.
 
12) When a patient is sensitized, precise characterization of HLA antibodies & complete HLA typing of the donor must be performed.
 
13) HLA antibody screening should be performed at a frequency that accommodates the likelihood of an imminent transplantation based on local waiting times & the immunologic risk of adverse outcome such as in
highly sensitized patients.
 
14) A minimum of 2 sera obtained at different time points should be tested to confirm presence or absence of HLA antibodies.
 
15) Sera should be tested after known sensitizing events, proinflammatory events, & at regular intervals once listed for transplantation.
 
16) Kidney
 
a. Unacceptable HLA antigens should be a part of kidney allocation algorithms.
 
b. Accurate XM prediction depends on complete HLA typing.
To minimize the incidence of unexpected positive XM in paired exchange registries, the donor should be typed at HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLADRB4, HLA-DRB5, HLA-DQA, HLA-DQB, HLA-DPA, & HLA-DPB loci.
 
c. A renal transplantation can be performed without a prospective CDC or flow XM if SAB testing indicates the consistent absence of DSA against HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLADRB4, HLA-DRB5, HLA-DQA, HLA-DQB, HLA-DPA, & HLA-DPB locus antigens. Each center needs to develop its policy in agreement with regulatory bodies & clinical programs.
 
d. Risk assessment should include HLA antibody specificities identified in historic sera.
 
e. In renal transplantation, if DSA is present but the CDC XM against donor T & B cells is negative, this should be regarded as an increased immunologic risk but not necessarily a contraindication to transplantation, especially
after elimination of DSA by desensitization.
f. To optimize access to transplantation of highly sensitized recipients, inclusion of patients in special programs, such as kidney paired donation, AM, or desensitization, should be considered.
 
g. HLAmatching should be part of the allocation procedures to reduce the probability of developing HLA antibody, rejection, & graft loss.
 
h. ABOi is no longer an absolute contraindication in kidney transplantation & ABOi transplants can be successfully performed in recipients in whom isoagglutinin titers have been lowered to acceptable levels.
 
i. Based on current evidence, no recommendation can be made for routine pretransplantation testing for non-HLA antibodies other than ABO.
 
17) Heart
 
a. Determination  of DSA must be performed because it is critical to improve short-term outcomes & preventing early acute rejection.
 
b. Desensitization therapy should be considered in sensitized heart transplant recipients.
 
18) Lung
 
a.DSA in recent serum should be avoided in lung transplantation if possible.
 
19) Liver
 
a. The liver allograft may be partially resistant to antibody mediated
damage; however, high-level DSA antibody may be associated with inferior outcomes & should be considered as a risk factor for graft dysfunction.
 
b. Screening for HLA antibodies is recommended in liver transplant recipients for risk stratification.
 
c. Donor tissue should be collected and stored in liver transplantation.
 
d. An XM should be performed in sensitized liver transplant recipients.
 
e. In sensitized recipients of combined liver-kidney transplantation,
the liver may not confer full protection for preventing AMR in the kidney & should be included in risk assessment.
 
20) Pancreas      
 
a. Recommendations for kidney transplantation should apply to the pancreas for SPK transplantation.
 
b. Pancreas is at risk for AMR & pretransplantation DSA should be avoided whenever possible.
 
c. In pancreas transplantation, AMR should be considered in the differential diagnosis of early graft thrombosis graft dysfunction.
 
21) Intestine
 
a. In intestinal transplantation, pretransplantation HLA antibodies should be determined. The risk assessment should be based on the level of DSA.
 
22) Islets
a. Based on the available literature, pretransplantation DSA are associated with impaired islet cells function posttransplantation & should be avoided.
 
Posttransplantation Group
 
23) Pretransplantation
 
a. DNA must be available on all donors for identification of donor antigens. This is essential for accurate DSA assessment.
 
b. Store frozen pretransplantation serum from recipients. The most current serum is acceptable; day of transplant serum recommended.
 
24) Posttransplantation (months 0-12)
a. Very high risk patients (desensitized): These patients are recognized to be at high risk for AMR & as such are treated with a desensitization protocol. Such protocols are not standardized & are center specific. Monitor DSA & conduct protocol biopsies in the first 3 months after transplantation:
 
1. If there is evidence of clinical or subclinical AMR, the patient should be treated. Efficacy of treatment is reflected as normal graft function & is associated with a reduction of DSA levels.
2. If there is a rapidly increasing level of DSA accompanied by a biopsy showing no rejection, initiation of therapy to reduce the DSA levels should be considered.
 
b. High-risk patients (DSA positive/XM negative): These patients are recognized to be at risk for early clinical or subclinical AMR. Monitor DSA & conduct a protocol biopsy in the first 3 months after transplantation.
 
1. If biopsy is positive for AMR, the objective is to treat. Efficacy of treatment is reflected as normal graft function & is associated with a reduction of DSA levels.
 
2. If there is a rapidly increasing level of DSA accompanied by a biopsy showing no rejection, initiation of therapy to reduce the DSA levels should be considered.
 
3. DSA persists in the absence of biopsy proven rejection, immunosuppression should not be reduced & additional monitoring should be considered.
 
4. If the DSA & biopsy are negative, follow as if low risk (see d.1.) unless there is an inflammatory event, in which case additional monitoring for DSA is recommended.
 
c. Intermediate-risk patients: Includes history of sensitization to donor antigen(s) by CDC & SPI but currently negative & history of sensitization with at least one positive test for HLA antibodies. Monitor for DSA
within the 1st  month.
1. If a DSA present, then perform a biopsy. A biopsy is recommended because of published data that document an association between DSA & clinical or subclinical rejection.
2. If biopsy is positive for rejection, the objective is to treat. Efficacy of treatment is reflected as normal graft function & is associated with a reduction of DSA levels.
3. In the absence of biopsy-proven rejection, additional DSA monitoring should be considered within the first year.
4. Patients with a DSA in the absence of biopsy-proven rejection should not be considered for reduction in immunosuppression.
5. In the absence of a DSA follow-up as if low risk (see d.1.).
d. Low-risk patients (nonsensitized, first transplantation)
1. Screen for DSA under the following circumstances:
a) at least once 3 to 12 months after transplantation.
b) whenever significant change in maintenance immunosuppression
is considered (e.g., minimization/withdrawal/conversion).
c) suspected nonadherence.
d) graft dysfunction.
e) before transfer of care to a remote center outside the transplant center.
2. If DSA present, then perform a biopsy. A biopsy is recommended because of published data that document an association between DSA & clinical or subclinical rejection.
3. If the biopsy is positive for rejection the objective is to treat. Efficacy of treatment is reflected as normal graft function & is associated with a reduction of DSA levels.
4. In the absence of biopsy-proven rejection additional DSA, monitoring should be considered within the first year.
5. Patients with a DSA in the absence of biopsy-proven rejection should not be considered for reduction in immunosuppression.
6. If no DSA present, then no additional testing in the first year is recommended in the absence of circumstances listed under point 1 above.
 
25) Posttransplantation (month 12 onward)-applies to all risk categories
a. Store at least one serum sample per year (i.e., on the transplantation anniversary).
b. Evaluate DSA in a current serum if any of the following conditions occur:
1. Significant change in maintenance immunosuppression is considered (e.g., minimization/withdrawal/conversion).
2. Suspected nonadherence.
3. Graft dysfunction.
4. Before transfer of care to a remote center outside the transplant center.
c. If de novo DSA present or if there is an increase in previous DSA levels, perform a biopsy. A biopsy is recommended because of published data that document an association between DSA and clinical or subclinical rejection.
1. If biopsy is positive for AMR, the objective is to treat.
Efficacy of treatment is reflected as normal graft function and is associated with a reduction of DSA levels.
2. If biopsy is negative (no sign of rejection) monitor the DSA and monitor for a change in graft function.
3. Patients with a DSA even without biopsy proven rejection should not be considered for reduction in immunosuppression.
 
Technical Group
1. A coordinated search for a selected bank of HLA reference antibodies to be used for assessing the interlaboratory variability in SPI antibody testing should be undertaken. Antibodies to MICA & relevant non-HLA antigens should be included.
2. The C1q modified SAB antibody identification requires more research & validation to understand its application to risk assessment & monitoring efficacy of treatment.
3. If production of the SPI donor-specific XM kit is continued, further research will be required to establish the clinical role of such a test.
4. The clinical endothelial cell XM for the detection of antiendothelial antibodies needs further investigation.
5. SPI for the detection of non-HLA anti-endothelial antibodies require development.
6. Multicenter studies are required to establish the clinical utility of testing for antibodies to tissue-specific non-HLA antigens such as the angiotensin II type 1 receptor.
7. The role of antibodies to epitopes found on HLA-E merits further investigation.
8. The short-term & long-term clinical effect of low levels of HLA antibodies detected by SPI requires further investigation.
Pretransplantation Group
9. More systematic studies for immunologic risk assessment that include long-term outcome data are required.
10. Randomized controlled studies to analyze the efficacy & safety of different desensitization protocols are required.
11. Additional studies are needed to determine the role of preexisting complement fixing versus noncomplement fixing antibodies to HLA& their role in organ transplantation.
12. Efficacy of desensitization in achieving good long-term graft
survival needs to be established in heart transplantation.
13. Efficacy of desensitization in achieving good long-term graft survival needs to be established in lung transplantation.
14. More systematic studies are required on the impact of DSA on pancreas transplant alone or in combination with a kidney.
15. Detailed analysis is required to establish the impact of pre-transplantation DSA on outcomes in intestine & multivisceral transplantation.
16. Further studies are needed to determine the risk of preexisting
DSA in islet cell transplantation.
17. Standardization of the methodology for determining ABO isoagglutinin titers is required.
18. Future studies are required to define the role of preexisting antibodies to non-HLA & self- antigen on the outcome of solid organ & cellular transplantations.
 
Posttransplantation Group
A need is recognized for the following:
19. Serial screening of serum to determine timing of onset of de novo DSA before onset of graft dysfunction.
20. Protocol biopsies at first appearance of de novo DSA to document pathologic correlation.
21. Assessment of DSA for complement fixing activity & correlation with clinical events (e.g., DSA C1q binding& IgG subclass specificity of DSA).
22. Clinical trials that include serial DSA monitoring posttreatment & posttreatment biopsies to correlate DSA levels with histologic response to therapy & long-term outcome.
23. Clinical trials to prevent production of DSA.
Future Directions
Although DSA are routinely found in patients who experience immunologic graft loss, whether the antibodies are causal or a consequence of other process(es) is still not well unanswered. So, it has been a challenge to know what, if anything to do when antibodies are identified posttransplantation.
 
Reemergence  of DSA that were present immediately pretransplantation, which required the recipient to be desensitized in order for the patient to be transplanted, requires an immediate response to try & prevent a catastrophic event.
 
The response of any given patient to therapy ranges from complete elimination of DSA & the accompanying symptoms of graft rejection to a failure to eliminate or even reduce the DSA while the patient steadily proceeds to graft rejection.
 
Studies indicated that certain patients do very poorly posttransplantation even when the antibodies appear to be completely eliminated after desensitization & therefore should not be entered into a desensitization program. 30% of patients who undergo desensitization still experience AMR & are at risk to develop TG & ultimately graft loss.
 
There  is no predictive test to identify into which category (the 70% of patients who do well or the 30% who experience AMR) the patient will fall.
 
Newly developed tests aimed at detecting & quantifying memory B cells & plasma cells that produce DSA may eventually help to categorize a patient’s posttransplantation risk to have transplant threatening DSA reappear.
 
Until these tests become reliable, the risk of any given desensitized patient cannot be accurately assessed.
Experience  indicates that 15% to 20% of patients will develop de novo DSA posttransplantation.
 
How frequently posttransplantation candidates without pretransplantation DSA should be monitored & what to do if antibodies are identified remains to be answered,
Can early intervention prevent graft loss due to AMR?
Multicenter  randomized control trials ared needed to answer this question.

• How would this study change your practice?

The study touched important points regarding many differences between the relatively new SPI technology & the older methods, which are still being used in low resources centers like ours.
In our center we even don’t have a Luminex.
We send samples for PRA to another center in a city 180 km far; it is the only center countrywide.
We  use CDC XM for most of our patients. Flow-cytometric XM is done only in highly sensitized patients.
We do HLA typing for HLA-A,B, & DR classes; by PCR using  SSP.
Inspite of this, the information from this study will hopefully help us to shape our future and try to make some changes despite to our limited resources.