**3.2 HLA typing**

HLA typing is performed to assess HLA mismatches between the donor and recipient. Preformed DSA are also measured at this stage. HLA mismatch exists when an HLA antigen is present in the donor but not in the recipient. The higher the mismatches, the more foreign the graft is immunologically and is likely to mount an immune response.

Historically, typing has focused on HLA-A, HLA-B, HLA-DQ, and HLA-DR loci in renal transplantation. The reason for emphasizing on these loci was that the DSAs against these antigens pose a significant risk of hyperacute and accelerated rejection; however, typing limitations also had a role.

Serological assays were used to type HLA initially. However, it is now replaced by DNA-based molecular techniques, which include sequence-specific priming (SSP), real-time PCR, and reverse sequence-specific oligonucleotide probing (rSSOP).

### **3.3 What is HLA sensitization?**

The presence of HLA-specific antibodies in an individual's serum defines HLA sensitization. HLA antibodies directed against the donor HLA are called donor specific antibodies (DSA). Sensitization occurs through blood transfusion, pregnancy, and transplantation. **Table 2** illustrates the variation in the vigor of the alloimmune response to the type of sensitizing event [9, 10]. Proinflammatory conditions like infections and trauma influence the strength and breadth of the HLA antibodies [11].

Positive complement-dependent crossmatch (CDCXM) or flow cytometer crossmatch (FCXM) before transplantation defines HLAi transplantation. Class I DSA is associated with acute rejection, while class II DSA causes chronic rejection [12, 13].

## **3.4 DSA screening and cross match**

DSA screening is performed using cell-based crossmatch and solid phase techniques. Due to the limitations of the cell-based assays, most HLA laboratories have moved to more advanced assays.


**Table 2.**

*HLA seroconversion rates after various sensitization events.*

Solid phase multiplex assays have significantly improved the detection of anti-HLA DSA. It is performed by incubating the recipient's serum with the polystyrene beads to which purified HLA antigens are attached. Then, a florescent conjugated IgG antibody is added. Flow cytometer or Luminex reads the test results.

Most laboratories will first perform screening using beads with multiple HLA antigens. If the screening test is positive, then a single antigen bead (SAB) assay is performed to detect the specificity of the antibody. SAB assay is a semiquantitative measure and can identify only anti-HLA IgG DSA. The output of the assay is expressed as mean or median fluorescence intensity (MFI). It represents the strength and amount of the DSA. A standardized cut-off MFI value above which the result is reported positive is not clearly defined. Each laboratory sets its cut-off point. A study reported >90% consensus among the HLA laboratories for MFI positive cut-off range of 1000–1500 [14]. MFI > 5000 is considered cytotoxic by most.

Despite the improved sensitivity of solid-phase technology, it has certain limitations and cell-based assays are still in use. False positive results can occur due to denatured proteins on the beads. C1q and complement products could bind to the beads, thus obscuring antibody detection (prozone effect) [15]. Similarly, IgM antibodies or intravenous immunoglobulins could hinder IgG binding to the beads, thus giving a false negative result. This effect could be countered by using ethylene diamine tetraacetic acid (EDTA), dithiothreitol (DDT), or heat inactivation [16, 17]. Antibodies against public epitopes bind with more than one bead, causing an underestimation of the true DSA. Renal transplant candidates with no history of sensitization and little or no DSA detection on solid phase assay do not require further investigations.

Due to technical reasons, many laboratories are transitioning away from cell-based crossmatches. However, despite this shift, it is still performed in specific situations. Two types of cell-based cross matches are available.

Complement-dependent cytotoxicity crossmatch (CDCXM) assay is performed by mixing the recipient's serum with donor lymphocytes (T and B). Then, an exogenous complement and a viability dye are added. Suppose a complement binding DSA is present in the recipient's serum. In that case, it will bind to the HLA antigens on the donor lymphocyte, which activates complement and causes cell lysis, rendering the test positive. A positive T cell CDCXM identifies class I antibodies, and a positive B cell crossmatch indicates antibodies against class I and II antigens.

Flow cytometry crossmatch (FCXM) assay is more sensitive and detects IgG DSA regardless of its ability to activate complement [18]. Like CDCXM, FCXM is performed by mixing the donor's lymphocytes with the recipient's sera. The flow cytometer reads the analysis results and expresses them as the median fluorescence index (MFI). The presence of DSA is determined by channel shift of fluorescence intensity beyond a pre-determined value.

Cell-based crossmatch techniques require live donor cells and have low precision in detecting the specificity of anti-HLA antibodies. Non-HLA antibodies (including autoimmune antibodies) can give false positive results. Therefore, an autologous crossmatch is performed when a cell-based crossmatch assay is positive to evaluate the presence of autoantibodies. Low titer DSA may be unable to activate the complement pathway and missed. This deficiency could be improved by using anti-human globulin (AHG) which, amplifies complement activation. AHG also helps in differentiating complement and non-complement binding DSAs. IgM HLA and non-HLA antibodies can cause false positive crossmatch. Cell-based assays are not standardized, and therefore, inter-laboratory variations exist. Furthermore, these assays are time-consuming prolonging cold ischemia time and affect organ sharing [19].

*Desensitization in Solid Organ Transplantation DOI: http://dx.doi.org/10.5772/intechopen.113262*

A tool called panel reactive antibodies (PRA) measures the degree of sensitization. Previously, PRA was determined using cell-based assays; however, United Network for Organ Sharing (UNOS) mandated to replace it with calculated PRA (cPRA) in 2007. cPRA is based on SAB assays. This tool provides HLA antibody specificity in combination with HLA antigen frequencies in the donor population. For example, a patient with DSA against HLA B44 (which is reported in 27% of the United States donors), will have 27% cPRA. Put differently, this patient will not be able to get organ from 27% of the donors.

The degree of sensitization is not precisely defined; however, most centers consider cPRA of 0–10% non-sensitized and >80% highly sensitized.

Virtual crossmatch (VCXM) provides valuable information about the risk of antibody-mediated rejection (ABMR) using the recipient's DSA against the donor HLA typing without running an actual crossmatch. Virtual crossmatch (VCXM) is considered positive when a DSA occurs at a cut-off value above the acceptable or manageable level [19]. Virtual crossmatch accurately predicts the outcomes of FCXM in >85% of the cases [20], but the correlation with CDCXM is low due to the higher sensitivity of solid phase assays. The negative predictive value of VCXM is high for early ABMR. In sensitized patients, the error rate of VCXM is 15%; therefore, actual crossmatch is still required for risk stratification. VCXM has shortened timelines for organ allocation in heart, lung, and kidney transplantation [19, 21]. Actual crossmatch could be omitted in non-sensitized recipients with no history of sensitization and negative VCXM. An incomplete donor HLA profile diminishes the accuracy of virtual crossmatch.

#### **3.5 Integration of immune data for risk assessment**

Integrating data from multiple immunologic tests provides a better idea of the risk, as illustrated in **Table 3**.

### **4. Outcomes of desensitization in HLAi renal transplantation**

#### **4.1 Patient and graft survival**

The outcome data of HLAi renal transplantation is contradictory. Two retrospective studies from the United States compared patient survival after HLAi renal transplant to waitlist and transplanted population. **Table 4** illustrates the results.

In contrast to the above data, a retrospective study of HLAi renal transplantation from the UK did not show a significant difference in patient survival compared to those who continued dialysis while waiting for transplant or got transplanted (p = 0.98) and waitlisted only (p = 0.44) [22]. Controls were matched for age, sex, cPRA, blood group, diabetes mellitus as the primary cause of ESRD, number of previous transplants, duration on paired kidney donation list, and ESRD vintage. Positive CDCXM or FCXM defined HLA incompatibility. The author excluded patients with negative crossmatch but positive DSA.

Another study, including 326 highly sensitized patients (>80% PRA), examined patient survival [23]. Thirty-six patients were desensitized with rituximab and plasmapheresis. Thirty patients received kidney transplants. One hundred forty-nine patients were transplanted without desensitization, and 141 remained on dialysis. The investigators did not observe significant differences in mortality among the groups (HR = 0.48, p = 0.22).


#### **Table 3.**

*Immunological risk assessment in renal transplant candidate.*


#### **Table 4.**

*Patient survival post renal transplant with desensitization.*

*Desensitization in Solid Organ Transplantation DOI: http://dx.doi.org/10.5772/intechopen.113262*

Another study, including 372 desensitized recipients, showed no difference in patient or graft survival over 60 months among the study groups [24].

The reasons for contradictory results between the United States and Europe are unknown. However, the survival rate on dialysis therapy in the United States is comparatively lower.

**Table 5** summarizes graft and patient outcome data from other studies.

#### **4.2 Acute rejection**

The risk of acute rejection is higher in HLAi renal transplants than in HLAcompatible, living donor transplants (21 vs. 8%) [36]. The risk increases proportionality with the degree of incompatibility. Acute rejection occurred in 18% of patients with DSA but negative FCXM, 21% with positive FCXM but negative CDCXM, and 22% with positive CDCXM. Interestingly, most of the rejections were ABMR [24].

#### **4.3 Infectious complications**

The data about the risk of infection after desensitization is contradictory. A retrospective study compared infections in HLAi and ABOi to compatible transplant recipients. There was no significant difference in the infection rates between the groups after 18 months [25]. On the contrary, a Korean organ Transplant registry study reported a higher rate of infections in the HLAi and ABOi transplantation (89 vs. 27%) [26].

A European study reported a higher rate of deaths from infection in the first year after ABOi transplants [27].


#### **Table 5.**

*Graft and patient survival after HLAi transplantation.*

Data about malignancy risk in HLAi renal transplants is limited. The risk of malignancy in ABOi renal transplants is like that of ABO-compatible transplants.

#### **4.4 Cost of HLAi Tx**

An HLAi renal transplant costs more than an HLA-compatible transplant due to pre-transplant conditioning, longer hospitalization, monitoring, and frequent biopsies. The mean cost of a HLAi renal transplant was \$151,024 compared to \$106,306 in a matched control study [28]. The price increases incrementally with the degree of incompatibility.

Prospective studies are required to analyze the quality of life and morbidity associated with desensitization.
