Biomarkers and a tailored approach for immune monitoring in kidney transplantation

Table 1 Recommendations and suggestions on the incorporation of biomarkers and surveillance biopsies in kidney transplantation

Scenario A: Patients with acute kidney transplant dysfunction on whom a kidney transplant biopsy has been performed to exclude rejection
Recommendations
A1	Diagnose rejection if present in kidney transplant biopsies according to the Banff classification (using the most current update; now the 2015 update), and report it in a systematic way
A2	Quantify BK viremiaa and BK virus (BKV) nephropathy by specific staining
A3	Detect anti-HLA antibodies/DSAd and define their immunoglobulin class, complement fixing capacities and titres through dilutions
Suggestions
A4	Bank serum, plasma, urine, peripheral blood mononuclear cells (PBMC) and kidney transplant tissue for future biomarker researchc
A5	Exclude active infection by cytomegalovirus (CMV) and Epstein-Barr virus (EBV)a
A6	Generate a data base with detailed clinical and immunological variables, ideally, using a standardized data base from a consortium or a large multicentre/multinational collaboration
A7	Test any experimental biomarker(s) of your choice and correlate it/them with standard clinical variables and a detailed immune profile. The use of validated disease classifiers and archetypes appears to have more diagnostic accuracy than the use of single biomarkers
A8	Perform a surveillance biopsy if kidney function and other clinical or laboratory parameters do not improve as expected after treatment to exclude persisting rejection or transformation to another type of rejectionb
Scenario B: Patients with acute kidney transplant dysfunction on whom a kidney transplant biopsy is being considered to exclude rejection
Recommendations
B1	Quantify BK viremiaa
B2	Detect anti-HLA antibodies/DSAd and define their immunoglobulin class, complement fixing capacities and titres through dilutions; and perform a kidney transplant biopsy if DSA are detected
B3	Use validated disease classifiers and archetypes (if available) to enhance to pre-test probability for rejection, and perform a kidney transplant biopsy if positive
B4	If a kidney transplant biopsy is performed, consider the recommendations and suggestions for Scenario A
Suggestions
B4	Bank serum, plasma, urine and PBMC for future biomarker researchc
B5	Exclude CMV and EBV infectiona
B6	Generate a data base with detailed clinical and immunological variables, ideally, using a standardized data base from a consortium or a large multicentre/multinational collaboration
B7	Test any experimental biomarker(s) of your choice and correlate it/them with standard clinical variables and a detailed immune profile. The use of validated disease classifiers and archetypes appears to have more diagnostic accuracy than the use of single biomarkers
Scenario C: Patients with: (1) stable kidney function; (2) low immunological risk for ABMR with lack of preformed DSA; and (3) low immunological risk for TCMR or for the synthesis of de novo DSA due to no or low degree of HLA mismatch[16-18]
Recommendations
C1	Detect anti-HLA antibodies/DSAd after a sensitization event (transfusions, pregnancies or other transplants e.g., pancreas after kidney transplantation) and define their immunoglobulin class, complement fixing capacities and titres through dilutions
C2	Perform a kidney transplant biopsy if DSA are detected, diagnose it according to the Banff classification 2015 update and exclude intra-graft BKV infection by specific staining
C3	In case of kidney dysfunction, consider the recommendations and suggestions for Scenarios A or B
Suggestions
C4	Test any experimental biomarker(s) of your choice at pre-selected time points and correlate it/them with standard clinical variables and a detailed immune profile. Select time points based on the modal distribution of rejection in a specific population of patients with similar immunological risk, ideally derived from your own registry
C5	Consider surveillance biopsies that exclude subclinical rejection and banking of kidney transplant tissue for biomarker researchc. Recommendation to select time points based on the modal distribution of rejection in a specific population of patients with similar immunological risk, ideally derived from your own registry
C6	Detect anti-HLA antibodies/DSAd at your pre-selected time points, to define their immunoglobulin class, complement fixing capacities and titres through dilutions, and correlate them with standard clinical variables and a detailed immune profile. Select time points based on the modal distribution of rejection in a specific population of patients with similar immunological risk, ideally derived from your own registry. There are published consensus guidelines[19], but their recommendations are relatively arbitrary as well
C7	Bank serum, plasma, urine and PBMC at your pre-selected sampling time points and when kidney biopsies are performedc
C8	Exclude CMV and EBV infectiona
C9	Perform a biomarker-driven biopsy if your chosen validated biomarker for rejection (or any other anomaly) turns positive, and bank tissue for further biomarker research
Scenario D: Patients with: (1) stable kidney function; and (2) high immunological risk for ABMR due to preformed DSA (desensitized or not)
Recommendations
D1	Ensure adequate levels of immunosuppression and prevent non-compliance with treatmente
D2	Perform surveillance biopsies to exclude subclinical rejection and banking of kidney transplant tissue for biomarker researchc. Select time points based on the modal distribution of rejection in a specific population of patients with similar immunological risk, ideally derived from your own registry, but available guidelines[19] recommend them within the first 3 (or 6) mo post-transplantation
D3	Monitor anti-HLA antibodies/DSAd and define their immunoglobulin class, complement fixing capacities and titres through dilutions at your pre-selected time points and correlate them with standard clinical variables and a detailed immune profile. Select time points based on the modal distribution of rejection in a specific population of patients with similar immunological risk, ideally derived from your own registry; although there are published consensus guidelines[19]
D4	Detect anti-HLA antibodies/DSAd after a sensitization event (transfusions, pregnancies or other transplants, e.g., pancreas after kidneytransplantation) and define their immunoglobulin class, complement fixing capacities and titres through dilutions
D5	Perform a kidney transplant biopsy if DSA are detected, to diagnose it according to the Banff classification 2015 update and exclude intra-graft BKV infection by specific staining
D6	Perform a biomarker-driven biopsy if your chosen validated biomarker for rejection (or any other anomaly) turns positive, and bank tissue for further biomarker research
D7	In case of kidney dysfunction, we recommend to perform a kidney transplant biopsy and to consider the recommendations and suggestions for Scenario A
Suggestions
D8	Test any experimental biomarker(s) of your choice at pre-selected time points and correlate it/them with standard clinical variables and a detailed immune profile. Select time points based on the modal distribution of rejection in a specific population of patients with similar immunological risk, ideally derived from your own registry
D9	Bank serum, plasma, urine and PBMC at your pre-selected sampling time points and when kidney biopsies are performedc
D10	Exclude CMV and EBV infectiona
Scenario E: Patients with: (1) stable kidney function; (2) high immunological risk for TCMR and for the synthesis of de novo DSA due to high degree HLA mismatch[16-18]; and (3) without preformed DSA
Recommendations
E1	Ensure adequate levels of immunosuppression and prevent non-compliance with treatmente
E2	Detect anti-HLA antibodies/DSAd, especially in those with HLA-B and HLA-DRB1 mismatches, thought to be more immunogenic[16], at your pre-selected time points and correlate them with standard clinical variables and a detailed immune profile. Define immunoglobulin class, complement fixing capacities and titres through dilutions. Select time points based on the modal distribution of rejection in a specific population of patients with similar immunological risk, ideally derived from your own registry, although there are published consensus guidelines[19]
E3	Detect anti-HLA antibodies/DSAd after a sensitization event (transfusions, pregnancies or other transplants, e.g., pancreas after kidneytransplantation) and define their immunoglobulin class, complement fixing capacities and titres through dilutions
E4	Perform a kidney transplant biopsy if DSA are detected, diagnose according to the Banff classification 2015 update and exclude intra-graft BKV infection by specific staining
E5	In case of kidney dysfunction, perform a kidney transplant biopsy, especially in those with HLA-B and HLA-DRB1 mismatches, thought to be more immunogenic, and consider the recommendations and suggestions for Scenario A
Suggestions
E6	Test any experimental biomarker(s) of your choice at pre-selected time points and correlate it/them with standard clinical variables and a detailed immune profile. Select time points based on the modal distribution of rejection in a specific population of patients with similar immunological risk, ideally derived from your own registry
E7	Suggest surveillance biopsies exclude subclinical rejection and banking of kidney transplant tissue for biomarker researchc. Select time points based on the modal distribution of rejection in a specific population of patients with similar immunological risk, ideally derived from your own registry
E8	Bank serum, plasma, urine and PBMC at your pre-selected sampling time points and when kidney biopsies are performedc
E9	Exclude CMV and EBV infectiona
E10	Perform a biomarker-driven biopsy if your chosen validated biomarker for rejection (or any other anomaly) turns positive, and bank tissue for further biomarker research
Scenario F: Patients with: (1) stable kidney function; (2) high immunological risk for ABMR due to preformed DSA; and (3) high immunological risk for TCMR and for the synthesis of de novo DSA due to high degree HLA mismatch[16-18]
Recommendation
F1	Follow our recommendations and suggestions for Scenarios D and E
Scenario G: Patients with delayed graft function (DGF)
Recommendations
G1	Perform a kidney transplant biopsy if DGF extends beyond the first week post-transplantation without an obvious explanation, and subsequently every 7-10 d if DGF persists[14]
G2	Detect anti-HLA antibodies/DSAd if DGF extends beyond the first week post-transplantation without an obvious explanation, and subsequently every 7-10 d if DGF persists, and define their immunoglobulin class, complement fixing capacities and titres through dilutions
G3	Perform a kidney transplant biopsy if DSA are detected, to diagnose it according to the Banff classification 2015 update and exclude intra-graft BKV infection by specific staining
Suggestions
G4	Define lower threshold for performing a kidney transplant biopsy in patients with DGF and pre-formed DSA or with HLA-B and HLA-DRB1 mismatches thought to be more immunogenic[16]
G5	Bank serum, plasma, urine and PBMC at the protocolised sampling time points and when kidney biopsies are performedc
G6	Bank kidney transplant tissue for biomarker research whenever a biopsy is performedc
G7	Test any experimental biomarker(s) of your choice at protocolised time points and correlate it/them with standard clinical variables and a detailed immune profilec
G8	Perform a biomarker-driven biopsy if your chosen validated biomarker for rejection (or any other anomaly) turns positive, and bank tissue for further biomarker research
G9	Exclude active CMV and EBV infectiona
Scenario H: Every kidney transplant patient included in a clinical trial
Recommendations
H1	Bank serum, plasma, urine and PBMC at the protocolised sampling time points and when kidney biopsies are performedcf
H2	Bank kidney transplant tissue for biomarker research whenever a biopsy is performedcf
H3	Test any experimental biomarker(s) of your choice at the sampling points established by the trial designers and correlate it/them with
H4	Consider performing surveillance biopsies at important assessment points as per trial protocol (which can help to exclude subclinical rejection and to assess histopathological response to interventions) and banking of kidney transplant tissue for biomarker researchc

^aThese infections can present with kidney dysfunction, trigger or appear around a rejection episode, but importantly viraemia, especially at high levels, will elicit cytotoxic-type and other immune responses that can interfere with the interpretation of biomarkers.

^bThis is another opportunity for biomarker testing, especially if its kinetics post-treatment are known or being tested.

^cWhen banking samples, we suggest to process them and store them with the vision that they could be analysed using different technologies (e.g., RNA- or proteomics-friendly sample processing), even if those technologies are not available in your lab, as the research world is developing towards more constructive collaborations and cross-validation approaches. In such way, laboratories will end up with legacy sample banks from highly characterized patients with several follow up times points, in which future technologies (pending improvements or not developed yet) could be easily applied, saving huge time to researchers (no further recruitment and sample acquisition), minimizing the risk of including patients to similar protocols (just because the technology has changed) and maximizing previous patients effort and kindness; at least for pilot, exploratory and cross-validation studies. Seek advice on how to maximise your sample banking from an experienced laboratory. Strict protocols should be devised and followed up and biobanking details of the samples should be recorded (time and date of collection, type of tube, type of anti-coagulant, additives for preservation, if centrifuged the speed of centrifugation in “g”, sample processor – if a person – or a machine, etc.). It is important to consider the easiness of the retrieval process of the data as it is inputted (any free text or absence of drop-down lists from choice answers will result in manual-dependent retrieval, which will be time consuming and expensive.

^dWe recommend high resolution tissue typing of HLA-A; -B; -C; -DP; -DQ; and -DRB1,3,4,5 alleles for both donor and recipient. This will ensure more accurate detection of anti-HLA DSA, and the use of algorithms to assess degree HLA mismatching like the HLAMatchmaker[17,18].

^eThis recommendation is important for every kidney transplant patient, but seems crucial for patients with augmented immunological risk.

^fFor clinical trials, we prefer to recommend rather than just suggest the inclusion of biomarker testing as the incorporation of biomarkers in diagnostic well-designed clinical trials is the best channel to validate biomarkers in a standardized controlled setting and maximize all the benefits from the trial.