Virtual Crossmatch in Kidney Transplantation

Shiva Samavat

Associate Professor of Nephrology

Labbafinejad Hospital

SBMU 2018.11.21

•All transplant candidates are screened to determine the degree of humoral sensitization to HLA antigens.

•Sensitization to HLA is of great concern in certain candidate populations: multiple blood transfusions, a previous kidney transplant, or from pregnancy.

2

3

4

bead

bead

Y

bead

YY

HLA HLA Antibody Detection Antibody

Pooled panel bead

Phenotype bead

Single antigen

bead

Application Screening Specification Specification

Relative Antigen density

Low Intermediate High

Antigen composition per bead

HLA I/IIHLA I or II phenotype

of one personSingle allele

antigen

Single Antigen Beads Could Help in:

• Calculated panel-reactive antibody (cPRA), which is presently the best estimate of likelihood of a positive XM/DSA to a randomly selected donor.

• Virtual crossmatching (VXM) to identify DSA pretransplant, in turn facilitating allocation and risk assessment.

• Detection of antibody against rare HLAs.

5

DSA & Positive Cross-match

Desensitization while on

waiting list

Approach to sensitized patients

Patients with a living donor Patients without living donor

Allocation System

cPRA; KDPI; EPTSUnacceptable antigen

Virtual Cross-match

No DSA & Negative Cross-match

Transplant

Desensitization Protocols

Paired Kidney Exchange

Virtual crossmatch

• The results of SPI testing can be used to predict the results of cell-based compatibility assays and are often referred to as a “virtual crossmatch” .

• “Virtual crossmatch” predicts “actual crossmatch” results.

• The rationale for moving to virtual crossmatching: •Use methods that mimic the sensitivity of the final crossmatch .•Reduce the time and cost for deceased donor work-up.•Deceased donor allocation for highly sensitized patients.

• It requires active communication between: •The HLA laboratory staff•Transplant coordinators•Transplant physicians

•Defining the HLA unacceptable antigens.•Methods for deceased donor HLA typing (the level of resolution).•Criteria to assess HLA compatibility.

Unacceptable antigen mismatches

• The determination of UAM is a critical decision step because:

•With increasing number of forbidden antigen, the patient’s chance to receive an organ offer diminishes dramatically.

• Conversely, unrecognized UAM frequently leads to inferior graft survival and futile organ shipments due to a positive crossmatch in the recipient center.

Most appropriate test and sensitivity level for the determination of UAM

Human Immunology. 2017; 78 (1): 19-23

• Depending on the algorithm, these patients had a vPRA between 60% and 71.5%, translating into a potential prolongation of waiting time between 1.5 and 1.8 years, respectively.

• An increase in vPRAs of 1% could be outweighed by an additional waiting time of 2.5 weeks.

•The decision which algorithm to use will remain a judgement call for HLA laboratories and transplant physicians and has to be balanced between an unknown prolongation of waiting time for a considerable number of potentially low risk patients and maximization of success rates for those patients transplanted.

•Defining the HLA unacceptable antigens.• Methods for deceased donor HLA typing (the level of resolution).•Criteria to assess HLA compatibility.

HLA Typing of Donor

•Needs a rapid and accurate technique:• SSP; SSOP; RT-PCR• 2-digit vs 4-digit typing (DQB1*03 vs DQB1*03:01)

• Locus to be typed:• HLA-DQA, DPA, C are not routinely typed.

• In a highly sensitized recipient:• High resolution typing• HLA-DQA, DPA, C

Assessment of HLA compatibility

•SABs are used to define broad and allele specific antibodies.•Donor- recipient matching could be done at various levels:•Allele matching•Epitope matching•CREG matching

•Allele matching could be done by:•High-resolution (four-digit) typing.•Online allele frequency databases

•Given the likelihood of increased allele-specific antibodies in highly sensitized patients, it may be time to re-examine the need for mandated high resolution deceased donor typing.

http://www.allelefrequencies.net/

Out of a total of 6924 procured kidneys from the 22 OPOs, 520 (7.5%) were exported out of the DSA for hPRA recipients.

Of these 520 kidneys, 402 (77.3%) were transplanted into the intended recipient (IR), 100 (19.2%) were transplanted into URs, and 18 (3.5%) were discarded.

Negative XM results were confirmed prior to kidney export.

Pre-export virtual XM results only

96.2% of kidneys with pre-export tissue XM went into IRs, compared to 80.7% for those who had virtual XM only, and 54.2% who had no XM done prior to shipping

•Kidneys were more likely to be transplanted into the intended recipient when tissue XMs were done prior to export of the kidney.

•A seemingly compatible transplant may in fact be incompatible due to unidentified allele-specific antibodies.

•Many of these XM issues may be related to transplant centers not listing antigens at the DP and/or DQA locus.

• Both V-XM and FC-XM detect alloantibodies based on fixing to a solid phase (donor cells or beads), while CDC-XM and cFC-XM cover only alloantibodies that bind complement and result in cell lysis.

Transplant immunology, Vol 4, March 2017, Pages 17-21

Avoidance of donors with HLA antigens against whom C1q-binding antibodies were detected would have prevented all positive crossmatches due to HLA antibodies.

•Screening for complement-binding antibodies by SPI in patients on the waiting list may further improve the identification of those donor-recipient-combinations, in whom the CDC-crossmatch would become positive.• In patients with relatively low C1q-PRA, all C1q-positive antibodies could be assigned as UAG to safely avoid positive CDC-crossmatches.

DSA MFI had poor correlation with both T cell (R2 = 0.493) and B cell (R2 = 0.571) FCXM MCS.

VXM Limitations

• Issues of incomplete donor HLA genotype

•Technical factors specific to SPI:• Use of appropriate MFI cutoff values • False positive reactions against the microbeads • Non-native HLA epitopes • False negative SPI results due to inhibition by interfering

substances or prozone effects

Technical aspects of SPI underlie majority of VCXM failures

•Anti-HLA antibodies not listed •Anti-HLA antibodies with

allelic specificity•Anti-HLADP antibodies,• False negative SPI results

(including antibodies against non-HLA antigens).

6.6 ± 6.7 new anti-HLA antibodies/sample at 1:10 serum dilution

Prozone effect false negative SPI results limit VCXM

Cumulative effects of multiple low-MFI DSA

• Multiple low-titer DSAs can have additive or synergistic effects and result in positive CXM.

• Ability of VCXM using sum MFI of all DSA to predict positive FCXM when VCXM based on single maximum DSA MFI did not predict positive FCXM.

Standard SPI does not detect all alloantibodies present capable of causing positive FCXM

IgM Anti-HLA Antibodies

Anti HLA class I Reactive Antigens IgG 

High Risk Antigens(MFI >1000)

None

Moderate Risk Antigens (MFI 500-1000)

None

 

Anti HLA class I Reactive Antigens IgM 

High Risk Antigens(MFI >1000)

A*23:01- A*24:02- A*25:01- A*26:01- A*34:01- A*68:01- B*07:02- B*08:01- B*13:01- B*14:02- B*15:01- B*18:01- B*27:05- B*38:01- B:40:01- B*44:02 - B*45:01- B*49:01- B*51:01- B*52:01- B*57:01

Moderate Risk Antigens (MFI 500-1000)

A*02:01- A*11:01- A*32:01

 Anti HLA class II Reactive Antigens IgG

 High Risk Antigens(MFI >1000)

None

Moderate Risk Antigens (MFI 500-1000)

None

Reactive Antigens  

High Risk Antigens(MFI >1000)

DRB1*01:03- DRB1*04:05

Moderate Risk Antigens (MFI 500-1000)

DRB1*10:01- DRB1*16:01- DRB1*04:04- DQB1*04:02- DQB1*03:01- DQB1*03:02

Reasons for a False-positive Virtual Crossmatch

• HLA molecules might be denatured during the production process exposing ‘novel’ epitopes.

• The detected HLADSAs might be directed against epitopes of the HLA molecules, which are accessible on SAB but not in vivo , and thus are probably not pathogenic (i.e. epitopes in the proximity of the cell membrane)

Summary

•VCXM is an important and powerful tool for organ allocation, and is increasingly used by transplant programs as a primary means for prospective assessment of immunologic compatibility.

•SPI and VCXM are imperfect.

• Increases transplantation probability in highly sensitized patients.•Predicts actual crossmatch results•Avoidance of unnecessary organ shipment•Save time

•Selection of cutoff values •Precise HLA typing of donor•Determination of C1q binding antibodies•Sample dilution to overcome prozone effect•Extended HLA SPI • IgM antibodies

Virtual Crossmatch in Kidney Transplantation:

Is It Better Than Actual Crossmatch?

Thanks for Your Attention

• Anti-HLA antibodies were tested with Luminex single antigen.• The recipients and donors were typed for HLA-A, -B, -DRB1, -

DQB1, the donors were also typed for -C, by SSP DNA-typing. • Unacceptable antigens for the V-XM were considered : class I or

class II HLA antibodies with MFI > 1000-1500.• A V-XM was performed for all deceased donors aged <65 years• Before transplantation a final T and B lymphocyte CDC-XM:

going ahead if the result was negative.

During the study period a total of 57 kidney grafts were initially allocated, although only 52 transplantations went ahead as the CDC-XM was positive in 5 patients (negative predictive value of the V-XM: 91.3%)

• MFI >2000 are assigned as “unacceptable”• MFI: 1000 to 2000 are assigned as “watch”

• The false negative V-XMs were related with:• Failures in introduction of the data.•HLA antigens not initially considered to be

unacceptable (HLA antibodies in a single historical serum sample or HLA antibodies with a low MFI). • Possible presence of non-HLA antibodies.•DP antibodies were related with the development of

AMR

•Restrictive VCXM criteria ensuring immunologic compatibility could unnecessarily exclude potentially compatible transplants (particularly for highly-sensitized patients).•Less restrictive VCXM approaches have higher risk of unpredicted incompatibility and resulting organ redirection, discard, or rejection.

•A CDC-crossmatch prior to transplantation should be maintained in highly immunized patients: assignment of all C1q-binding antibodies as UAG would more or less prevent allocation of organs to recipients with extremely high vPRA.

• CDC-crossmatch in patients with a recent immunizing event or patients with strong suspicion of possibly clinically relevant Non-HLA antibodies. (serum drawn immediately before transplantation)

• It is important to note however that a cell-based XM could be negative in the context of an actual allele-specific donor antibody.•Weak DSA not resulting in positive cell-based XM may still

present a risk of antibody-mediated rejection post-transplant.

• Multiple low-titer DSAs can have additive or synergistic effects and result in positive CXM.

• 23 cases where max MFI < 3000 and sum MFI>= 3000 correlated with a positive FCXM.

• 21 cases of a negative FCXM in the setting of max MFI < 3000 and sum MF>= 3000.

• Negative FCXM with positive VCXM using sum MFI occurred about 3-times as frequently when the multiple DSA were restricted to class II HLA antigens as compared to the

• The most common approach to deal with a potential allele-specific antibody was to request donor cells to perform a prospective cell-based crossmatch (XM). • might not have been available in a timely manner• A physical XM can very easily result in a false negative reaction,

providing misleading information to those centers willing to transplant across DSA as long as the physical XM was negative.• Another factor that can contribute to false negative crossmatches is

when the physical XM is performed without pronase treatment of donor cells

• Acceptable antigens are defined by the lack of antibody reactivity in complement-dependent cytotoxicity assays using target cells mismatched for a single HLA antigen, or single antigen-expressing cell lines.

• It is commonly accepted to define all those HLA antibody specificities as UAM that are cytotoxic in cell-based complement-dependent cytotoxicity (CDC) assays

• The relevance of a positive SAB test result, however, is less clear as the positive predictive value of a DSA detected by Luminex testing for the occurrence of early AMR and graft loss in an individual patient is low.

• neither the prognostic value of different MFI levels nor specific MFI cut-offs for the segregation of patients at risk from those without have clearly been established so far.

• It has been recommended to define DSA that are positive in solid phase but negative in cell-based assays only as UAM if they can be explained by a patient's immunization history

• HLA were defined as UAM if HLA-Abs were found in CDC assays or if the Luminex assay revealed HLA-Abs directed against HLA from previously failed grafts.

• Because cadaver donors had been routinely typed for HLA-A, -B, -DR, and -DQB molecules by sequence-specific primer methods, we did not consider antiHLA-C, -DQA, and -DPB DSA in performing v-XM.

• When relevant, donors were typed for these molecules.

• The limited specificity with both of the XM techniques (CDC-XM, 74%; FC-XM, 79%) was due to the presence of “acceptable DSA” (MFI <5,000) present in some serum samples.

• In particular, patient sera contained “acceptable DSA” and/or anti-DQA/DPB DSA in 145 (29%) of the 507 a-XMs.

• From the frequency of UAM in the ET population, the so called virtual panel reactivity (vPRA) value is calculated which reflects the percentage of organ donors that are not eligible for the patient. • vPRA is allocation relevant, and dependent on the height of

the vPRA value, patients can receive up to 100 additional points during organ allocation via the ‘mismatch probability score’ in which blood group compatibility and donor and recipient HLA typings are also considered.

•Kidney transplant candidates on the ET waiting list receive organ offers only from UAM-negative donors during the organ allocation process, a procedure which is designated as virtual XM (vXM).• This measure minimizes the number of cases with a

prolonged cold ischemia time by avoiding organ shipment to another center because of a positive XM result at the recipient center.

Methods for determination of UAM

• Sensitive solid-phase assays must be used in addition to the CDC technique. The CDC technique detects complement fixing antibodies in high-titer sera.

• Sera of all patients are tested using the SABmethod for the presence of HLA class I and class II IgG antibodies, at least at the time of active listing of the patient on the waiting list.

• For the determination of UAM, B-cell-specific cytotoxic techniques should also be used under the usage of dithiothreitol (DTT) (B-CDC-PRA and B-CDC-PRA-DTT).

The following precautions should be taken in the praxis of single antigen solid-phase assays:• Minimize prozone effects.

• The patient’s own four-digit allele specificities should be excluded in patients in whom antibodies against own HLA antigens are detected.

• SAB testing is associated with technical problems, such as false-positive results, which mainly originate from reactions of patients’ sera with denatured antigens and variable antigen densities on the beads.

• After addition of complement components, such as C1q, complement-binding HLA antibodies can be determined in the single antigen solid-phase assay.

Criteria for determination of UAM

• There is general consensus that cytotoxic HLA antibodies should be avoided. Therefore, all HLA class I and class II IgG antibody specificities that were identified in the CDC testing with unseparated lymphocytes or isolated T or B lymphocytes are considered a contraindication to transplantation and must be registered as UAM.

• In female patients with former pregnancies, either the HLA alleles of their children or the HLA alleles of the children’s father should be determined to identify potential immunizing antigen mismatches.

• Especially under the following conditions, HLA antibody specificities that give positive reactions only in the SAB or in the bead-based multiple antigen test, but are negative in CDC and/or other solid-phase assays, such as bead-PRA, flow-PRA, ELISA-screen, or ELISA-PRA, should not be considered as UAM :• a. if the HLA antibody specificities cannot be supported by the immunization

history of the patient.• b. if they belong to the unspecific reaction patterns that were previously

published to be caused either by natural antibodies or denatured antigens in solid-phase assays.