**5. HLA antibody detection assays**

#### **5.1. Calculated panel reactive antibody (cPRA)**

In the complement-dependent cytotoxicity (CDC) assay, recipient serum is mixed with donor lymphocytes and complement is supplemented. Presence of DSAs is indicated by the appearance of cytotoxicity. The CDC crossmatch was principal technique for detecting DSAs in kidney transplant candidates till the mid-1980s [12, 15]. The panel reactive antibody (PRA) assay, a simple test that predicts the likelihood of a transplant candidate finding a HLA compatible donor (with a negative CDC crossmatch). This test involves treating a panel of cells derived from a pool of individuals representative of the local donor population with recipient serum and noting the percentage of cells that develop cytotoxicity. Therefore, a patient with a high PRA percentage can be predicted to be HLA incompatible with a majority of the potential donors. Consequently, such patients have prolonged wait times for transplantation and may die waiting for a compatible organ [25]. The emergence of more sensitive solid phase assays that employ HLA antigen-coated beads have revealed a greater prevalence of pre-sensitization to HLA in potential transplant recipients than was previously appreciated. Using these sensitive techniques of HLA antibody detection (in recipient serum) in conjunction with modern HLA typing methods (to determine donor HLA typing), we can now estimate a transplant candidate's calculated panel reactive antibody (cPRA). This is an alternative to standard PRA testing. Transplant centers can designate HLA antigens to which the patient has been sensitized as "unacceptable." The cPRA is computed from HLA antigen frequencies among kidney donors in the United States and represents the percentage of actual organ donors that express any "unacceptable" HLA antigens [25]. One of the key elements of the Organ Procurement and Transplantation Network's (OPTN) new Kidney Allocation System (KAS) introduced in December 2014 is to allocate additional points to waitlisted kidney transplant candidates based on a CPRA sliding scale [26]. The aim of this policy is to increase access to transplantation for sensitized candidates [27]. Soon after the new KAS came into effect, the proportion of transplants being performed in patients with CPRA >98% rose from 2.4 to 13.4% [28]. Therefore, the new KAS appears to be accomplishing the goal of equitable allocation of scarce deceased donor organs to highly sensitized patients who are biologically disadvantaged.

#### **5.2. HLA antibody detection assays**

**Figure 3.** Platelet fibrin thrombi in glomerular capillary loops (black arrow) due to acute thrombotic microangiopathy in a patient with acute antibody mediated rejection. Pathology slides courtesy: Dr. Ming Wu, Department of Pathology,

**Figure 4.** Immunofluorescence staining showing diffuse linear C4d positivity in peritubular capillaries (white arrows).

In the complement-dependent cytotoxicity (CDC) assay, recipient serum is mixed with donor lymphocytes and complement is supplemented. Presence of DSAs is indicated by the appearance of cytotoxicity. The CDC crossmatch was principal technique for detecting DSAs in kidney transplant candidates till the mid-1980s [12, 15]. The panel reactive antibody (PRA) assay, a simple test that predicts the likelihood of a transplant candidate finding a HLA compatible donor (with a negative CDC crossmatch). This test involves treating a panel of cells derived from a pool of individuals representative of the local donor population with recipient serum and noting the percentage of cells that develop cytotoxicity. Therefore, a patient with a high PRA percentage can be predicted to be HLA incompatible with a majority of the potential donors. Consequently, such patients have prolonged wait times for transplantation

Pathology slides courtesy: Dr. Ming Wu, Department of Pathology, NYU Langone Medical Center.

**5. HLA antibody detection assays**

**5.1. Calculated panel reactive antibody (cPRA)**

NYU Langone Medical Center.

380 Organ Donation and Transplantation - Current Status and Future Challenges

Techniques for detection of HLA antibodies in transplant candidates have evolved considerably since the 1960s when the traditional cell-based complement dependent cytotoxicity (CDC) assay was first developed. Modern flow-cytometry and solid phase assays are far more sensitive than the CDC crossmatch. In the CDC crossmatch, recipient serum is incubated with donor lymphocytes, along with complement. A positive crossmatch is said to occur when DSAs present in the recipient serum bind the corresponding antigens expressed on the surface of donor T or B lymphocytes and activate complement leading to cell lysis [29]. The flow cytometry crossmatch (FCXM) developed in the 1980s was shown to be more sensitive than the CDC crossmatch and can detect lower strength, but clinically significant antibodies that are imperceptible to the CDC crossmatch [30]. In the FCXM, like the CDC crossmatch, donorspecific anti-HLA antibodies, if present in the recipient serum, bind HLA molecules on the surface of donor T or B lymphocytes. The flow cytometry technique detects these HLA bound anti-HLA antibodies by means of secondary fluorescein-labelled antihuman IgG [29]. The advent of solid phase assays for HLA antibody screening in the 1990s has redefined what it means to be sensitized to HLA. There are two varieties of solid phase assays – enzyme linked immunosorbent assay (ELISA) based methods and single antigen bead based methods. Single antigen bead based assays may employ either a flow cytometry platform or a Luminex platform to detect HLA [31]. The Luminex platform employs fluorochrome impregnated microbeads that are coated with specific HLA molecules. Donor-specific anti-HLA antibodies, if present in the recipient serum, bind HLA molecules coated on the surface of the beads. The microbeads are then incubated with phycoerythrin (PE)-labeled anti-human IgG antibodies. The Luminex dual laser system identifies the specificity of the bound anti-HLA antibodies[Ref]. The Luminex based assay has now become the most popular method of HLA antibody detection, both due to its superior sensitivity, as well as its ability to identify the antigenic specificity of the detected HLA antibody [29].
