**4. Diagnosis of antibody mediated rejection**

improved quality of life [3]. However, allograft rejection remains a major impediment to the longevity of renal allografts. Recognition of donor antigens as "non-self" by the host immune system elicits humoral and cell mediated immune responses that if left unchecked result in

**Figure 1.** In AMR, DSAs bind to human leukocyte antigens on graft vascular endothelium. This is followed by activation of complement, membrane attack complex-mediated cellular injury and infiltration of mononuclear cells. Reproduced

Human leukocyte antigens (HLA) play a vital role in host defense against foreign pathogens and in immune surveillance of tumors. These antigens are encoded by the major histocompatibility complex (MHC) which is a family of genes that encompasses a 3.6 million base pair genomic region, 6p21, on the short arm of chromosome 6 [5]. The MHC complex is divided into three regions representing three classes of genes—classes I, II, and III. The HLA genes that are involved in the immune response belong to classes, I and II [6]. Class I antigens are expressed on all nucleated cells whereas class II antigens are expressed only on professional antigen presenting cells (APCs)—dendritic cells, B-cells, and macrophages [7]. In the setting of infection, pathogen derived foreign antigens are presented to T-cells as peptides on the surface of major histocompatibility complex (MHC) molecules expressed on the surface of APCs. The ensuing signaling cascade results in the activation, proliferation, and differentiation of naïve T lymphocytes into subtypes with distinct cytokine profiles. Type 1 helper T (Th1) cells drive the cellular immune response mediated by CD8+ cytotoxic T lymphocytes, and type 2 helper T (Th2) cells drive B-cell mediated humoral

The heterogeneity of human MHC molecules enables the immune system to protect us from a variety of foreign pathogens. However, in the context of transplantation between genetically distinct individuals, these MHC polymorphisms elicit immune responses that can result in rejection of the allograft [7]. Two pathophysiologically distinct flavors of renal allograft rejection are recognized—cell mediated, and antibody mediated rejection. Both these types of rejection can manifest as acute or chronic clinicopathologic variants. Acute cell mediated rejection is characterized by infiltration of the allograft by effector T cells resulting in the typical features such as tubulitis, interstitial inflammation and in more advanced

**2. Human leukocyte antigen (HLA) system and allograft rejection**

the destruction of the allograft (**Figure 1**).

with permission from Montgomery et al. [4].

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immune response [2].

cases, endothelial arteritis [2, 8].

The Banff classification schema has been used internationally for scoring and classifying kidney transplant pathology findings since its first iteration was published in 1993. However, earlier versions dealt with AMR in an imprecise manner. The development of more sophisticated methods of detection of DSAs by means of solid-phase assays together with the sensitivity and specificity of C4d staining in peritubular capillaries in identifying AMR paved the way for rigorous morphological classification of AMR [23]. The cornerstones for the diagnosis for AMR are (1) Histologic evidence of acute tissue injury; (2) Evidence of current/recent antibody interaction with vascular endothelium; (3) Serologic evidence of DSAs. The updated 2015 Banff classification system recognizes acute active AMR and chronic active AMR and outlines detailed criteria for the diagnosis of each (**Table 1**) [24].


Glomerular and/or peritubular capillary infiltration with polymorphonuclear leukocytes and/ or macrophages represents microvascular inflammation and is the classic histologic feature of acute tissue injury in acute AMR (**Figure 2**). However, intimal or transmural arteritis, acute TMA and ATN can also denote acute AMR (**Figure 3**). Splitting or double contouring of the GBM (transplant glomerulopathy) as well as severe multi-lamination of peritubular capillary basement membrane are histologic features of chronic AMR. Detection of inert complement split product, C4d in peritubular capillaries by IF or IHC indicates antibody interaction with vascular endothelium (**Figure 4**). However, recognizing that complement independent pathways may be involved in the etiopathogenesis of AMR, the Banff classification also sets forth certain criteria (listed in **Table 1**) that allow for the diagnosis of acute or chronic AMR in patients without detectable C4d staining. Demonstration of circulating DSAs is a pre-requisite for diagnosis of AMR. Noting that non-HLA DSAs may result in clinical and histopathologic findings indistinguishable from AMR, Banff criteria for serologic evidence of DSAs require

g-glomerulitis, ptc-peritubular capillaritis, v-vasculitis, cg-chronic glomerulopathy (transplant glomerulopathy)

**Table 1.** Criteria for antibody mediated rejection as outlined by the Banff 2015 Meeting Work Group [24].

1. Linear C4d staining in peritubular capillaries (C4d2 or C4d3 by IF on frozen sections, or

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2. g = 0, ptc = 0, cg = 0 (by light microscopy and by EM if available), v = 0; no TMA, no peritubular capillary basement membrane multilayering, no acute tubular injury (in the

3. No acute cell-mediated rejection (Banff 1997 type 1A or greater) or borderline changes

All three features must be present for diagnosis:

C4d >0 by IHC on paraffin sections

absence of another apparent cause for this)

C4d staining without evidence of rejection

detection of either DSAs directed against donor HLA or "other" antigens [24].

**Figure 2.** Black arrows show infiltrating polymorphonuclear leukocytes in glomerular capillary loops (glomerulitis) in a patient undergoing acute oliguric antibody mediated rejection. Yellow arrows point to demarginated polymorphonuclear leukocytes in peritubular capillaries (peritubular capillaritis). Pathology slides courtesy: Dr. Ming Wu, Department of

Pathology, NYU Langone Medical Center.


**Table 1.** Criteria for antibody mediated rejection as outlined by the Banff 2015 Meeting Work Group [24].

Acute/active AbMR All three features must be present for diagnosis. Biopsies showing histological features plus

• Acute thrombotic microangiopathy in the absence of any other cause • Acute tubular injury in the absence of any other apparent cause

negative, based on the following criteria:

• Intimal or transmural arteritis (v<sup>c</sup> > 0)

C4d >0 by IHC on paraffin sections)

not sufficient, and g must be ≥1

injury, if thoroughly validated

3. Serologic evidence of DSAs (HLA or other antigens)

arterial involvement but are not required

C4d >0 by IHC on paraffin sections)

3. Serologic evidence of DSAs (HLA or other antigens):

Chronic active ABMR All three features must be present for diagnosis. As with acute/active ABMR, biopsies

tis, and/or ptc<sup>b</sup> > 0)

378 Organ Donation and Transplantation - Current Status and Future Challenges

least one of the following:

DSA testing

evident by EM only

least one of the following:

dited DSA testing

evidence of current/recent antibody interaction with vascular endothelium or DSA, but not both, may be designated as suspicious for acute/active ABMR. Lesions may be clinically acute or smoldering or may be subclinical; it should be noted if the lesion is C4d-positive or C4d-

• Microvascular inflammation (g<sup>a</sup> > 0 in the absence of recurrent or *de novo* glomerulonephri-

2. Evidence of current/recent antibody interaction with vascular endothelium, including at

• Linear C4d staining in peritubular capillaries (C4d2 or C4d3 by IF on frozen sections or

• At least moderate microvascular inflammation ([g + ptc] ≥2), although in the presence of acute T-cell mediated rejection (TCMR), borderline infiltrate, or infection; ptc ≥2 alone is

• Increased expression of gene transcripts in the biopsy tissue indicative of endothelial

showing histological features plus evidence of current/recent antibody interaction with vascular endothelium or DSA, but not both, may be designated as suspicious, and it should be

1. Histologic evidence of chronic tissue injury, including one or more of the following: • TG (cgd > 0), if no evidence of chronic thrombotic microangiopathy; includes changes

• Severe peritubular capillary basement membrane multilayering (requires EM)c

• Arterial intimal fibrosis of new onset, excluding other causes; leukocytes within the sclerotic intima favor chronic ABMR if there is no prior history of biopsy-proven TCMR with

2. Evidence of current/recent antibody interaction with vascular endothelium, including at

• Linear C4d staining in peritubular capillaries (C4d2 or C4d3 by IF on frozen sections, or

• At least moderate microvascular inflammation ([g + ptc] ≥2), although in the presence of acute TCMR, borderline infiltrate, or infection, ptc ≥2 alone is not sufficient and g must be ≥1

• Biopsies suspicious for AMR on the basis of meeting criteria 1 and 2 should prompt expe-

noted if the lesion is C4d-positive or C4d-negative, based on the criteria listed:

Biopsies suspicious for AMR on the basis of meeting criteria 1 and 2 should prompt expedited

1. Histologic evidence of acute tissue injury, including one or more of the following:

Glomerular and/or peritubular capillary infiltration with polymorphonuclear leukocytes and/ or macrophages represents microvascular inflammation and is the classic histologic feature of acute tissue injury in acute AMR (**Figure 2**). However, intimal or transmural arteritis, acute TMA and ATN can also denote acute AMR (**Figure 3**). Splitting or double contouring of the GBM (transplant glomerulopathy) as well as severe multi-lamination of peritubular capillary basement membrane are histologic features of chronic AMR. Detection of inert complement split product, C4d in peritubular capillaries by IF or IHC indicates antibody interaction with vascular endothelium (**Figure 4**). However, recognizing that complement independent pathways may be involved in the etiopathogenesis of AMR, the Banff classification also sets forth certain criteria (listed in **Table 1**) that allow for the diagnosis of acute or chronic AMR in patients without detectable C4d staining. Demonstration of circulating DSAs is a pre-requisite for diagnosis of AMR. Noting that non-HLA DSAs may result in clinical and histopathologic findings indistinguishable from AMR, Banff criteria for serologic evidence of DSAs require detection of either DSAs directed against donor HLA or "other" antigens [24].

**Figure 2.** Black arrows show infiltrating polymorphonuclear leukocytes in glomerular capillary loops (glomerulitis) in a patient undergoing acute oliguric antibody mediated rejection. Yellow arrows point to demarginated polymorphonuclear leukocytes in peritubular capillaries (peritubular capillaritis). Pathology slides courtesy: Dr. Ming Wu, Department of Pathology, NYU Langone Medical Center.

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

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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 specific-

who are biologically disadvantaged.

**5.2. HLA antibody detection assays**

ity of the detected HLA antibody [29].

**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, NYU Langone Medical Center.

**Figure 4.** Immunofluorescence staining showing diffuse linear C4d positivity in peritubular capillaries (white arrows). Pathology slides courtesy: Dr. Ming Wu, Department of Pathology, NYU Langone Medical Center.
