Desensitization in Solid Organ Transplantation

Abstract

Solid organ transplantation (SOT) has revolutionized the management of end-stage organ disease. Human Leukocyte antigen (HLA) sensitization and ABO incompatibility (ABOi) pose formidable barrier to SOT. The risk of acute rejection is high. They wait longer for compatible organs than their counterparts do. Furthermore, the graft and patient survival are suboptimal in incompatible transplants. Access to SOT could be promoted in this population by prioritizing them to well-matched organs in the allocation system via acceptable mismatch or paired donation programs. If these strategies fail to achieve transplantation, desensitization could provide an alternative. Desensitization is a process that allows transplantation in highly sensitized and ABO incompatible donor and recipient. Researchers initially developed principles of desensitization for kidney transplantation and have subsequently applied them to other types of solid organ transplantation. Desensitization protocols vary by the transplant center, but most use combinations of apheresis, intravenous immunoglobulin (IVIG), and anti-CD20 monoclonal antibodies. The desensitization aims to ease the immunological détente by removing preformed donor-specific alloantibodies (DSA) and creating a favorable immune environment for the allograft. Desensitization caries risk; therefore, careful patient selection and close monitoring are essential to mitigate the risk of complications. Further work is required to enhance the outcomes of desensitization.

1. Introduction

Solid organ transplantation is the optimal treatment for selected patients with end-stage organ disorders. Two important immunologic barriers to SOT are HLA sensitization and ABO incompatibility. The options for transplant candidates with incompatible donors are prioritization to well-matched organs in the allocation system, such as acceptable mismatch and paired donation programs or desensitization. Experience from renal transplantation forms the basis of contemporary desensitization protocols in SOT. This chapter will discuss desensitization in HLA incompatible (HLAi) and ABO incompatible renal transplantation. A brief account of desensitization in cardiac and lung transplantation is also provided.

2. HLAi renal transplantation

Antibodies against HLA are critical immunologic barriers to successful transplantation. The formation of HLA antibodies before or after transplantation is associated with poor graft and patient outcomes [1].

In the United States, 35% of waitlisted renal transplant candidates are sensitized. Fifteen percent of them are highly sensitized with calculated panel reactive antibodies (cPRA) >80% [2]. HLA sensitization restricts the number of compatible donors, thus reducing the chances of finding an acceptable HLA phenotype donor. Currently, only 6.5% of highly sensitized patients (cPRA > 80%) receive renal transplants each year [3]. Data from the United Network of Organ Sharing (UNOS) show longer waiting time for transplantation in sensitized individuals [4] (Table 1). Furthermore, sensitization increases the risk of hyper-acute, accelerated, and chronic graft rejection post-transplantation.

cPRA level (%)	Renal transplant candidates waiting for >5 yrs (%)
<80	8.8
80–89	10.7
90–98	17
99	20.9

Table 1.

Association of cPRA level and waiting time to renal transplantation.

Highly sensitized individuals have three options to access renal transplantation, i.e., either,

• Wait till a negative crossmatch donor becomes available, but this may take years OR,

• Consider paired kidney donation, but the chances of finding an appropriate donor are comparatively low OR,

• Undergo HLA incompatible transplantation with desensitization.

Desensitization reduces the cPRA by reducing DSA levels and thus increases the chances of getting an organ from a compatible donor. Furthermore, it lowers the risk of hyperacute rejection post-transplantation.

3. Essential concepts and terminology of transplant immunology

3.1 What are human leucocyte antigens (HLA)?

HLA are glycoproteins expressed on the cell surface that differentiate between ‘self’ and ‘non-self’ antigens. The primary purpose of HLA is to combat pathogens [5], but it poses a crucial barrier to transplantation.

The human major histocompatibility complex (MHC) on the short arm of chromosome 6 encodes HLA. They are the most polymorphic genes in the human genome. There are currently 37,068 HLA and related alleles in the human genome, and it is growing [6]. Antigen presenting cells present foreign antigens via MHC proteins to the T cells, which switch on the host immune system.

MHC encodes two classes of antigens, i.e., class I, which includes HLA-A, B, and C antigenic clusters and class II antigens comprising HLA-DQ, DP, and DR clusters [7]. Class I antigens are expressed on nucleated cells, platelets, and red blood cells (in ∼15% of individuals). Class II antigens are expressed on antigen-presenting cells, e.g., macrophages, B cells, and dendritic cells. However, other cells, e.g., epithelial and endothelial cells, can express class II antigens during inflammatory states, e.g., infections [8].

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].

Sensitization event	HLA seroconversion rate	Median fluorescence index (MFI)
Previous transplantation	50–75%	14,164
Pregnancy	38.3%	4185
Blood transfusion	20–33%	5548

Table 2.

HLA seroconversion rates after various sensitization events.

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.

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].

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.

CDCXM		FCXM		HLA antibody type	Interpretation
B cell	T cell	B cell	T cell
Current tests
+	+	+	+	IgG class I DSA	High risk for HAR Tx absolute contraindicated
+	—	+	—	IgG class II DSA	High risk for accelerated/refractory ABMR and poor long-term outcomes Tx contraindicated in most centers
+	—	+	—	IgG weak class I DSA	High risk for accelerated/refractory ABMR and poor long-term outcomes Tx contraindicated in most centers
—	—	+	+	IgG class I DSA	High risk for accelerated/refractory ABMR and poor long-term outcomes Tx contraindicated in most centers
—	—	+	—	IgG class II DSA	Intermediate risk. Consider PKD or desensitization
Historical tests
+	+	+	+	IgG class I DSA	High risk
+	—	+	—	IgG class II DSA	High risk
+	—	+	—	IgG weak class I DSA	Intermediate risk. Consider PKD or desensitization
—	—	+	+	IgG class I DSA	Intermediate risk. Consider PKD or desensitization
—	—	+	—	IgG class II DSA	Intermediate risk. Consider PKD or desensitization
Current or historical
—	—	—	—	IgG class I or II DSA	Low risk
—	—	+	+	HLA antibody but no DSA	Low risk
+	+	+	+	Negative	Low risk.; Autoreactive non-HLA IgG/IgM

Table 3.

Immunological risk assessment in renal transplant candidate.

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.

Author	Study group	1 yr (%)	3 yr (%)	4 yr (%)	8 yr (%)
Montgomery et al. [15]	HLAi renal Tx	90.6	85.7	80.6	80.6
	Wait listed dialysis or transplantation	93.1	77	65.6	49.1
	Wait listed only	91.1	67.2	51.5	30.5
Orandi et al. [22]	HLAi renal Tx	95	91.7	86	76.5
	Wait listed dialysis or transplantation	94	83.6	74.4	62.9
	Wait listed only	89	72.7	59.2	43.9

Table 4.

Patient survival post renal transplant with desensitization.

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).

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.

Author	Desensitization protocol	n=	ABMR (%)	F/U period (yr)	Graft survival (%)	Patient survival (%)
Tyan et al. [25]	High dose IVIG	15	6	1	81.8	100
Montgomery et al. [26]	PP + low dose IVIG + Rituximab	211	NR	1	>90	90.6
Guthoff et al. [27]	PP + low dose IVIG + Eculizumab	16	NR	1	100	100
Thielki et al. [28]	PP + low dose IVIG	51	24	2	81	91
Jordan et al. [29]	High dose IVIG + Rituximab	76	NR	2	84	95
Al Meshari et al. [30]	PP + IVIG	124	4	2	96	98
Klein et al. [31]	PP or IA + Rituximab	23	22	2	100	100
Jin et al. [32]	PP + low dose IVIG + Rituximab	6	0	2.7	100	100
Kahwaji et al. [33]	High dose IVIG	79	8	3	87.1	97.5
Warren et al. [34]	PP + low dose IVIG + splenectomy/PP + high dose IVIG/high dose IVIG alone/no Tx	102	31	5	70.7	83.5
Bentall et al. [35]	PP + low dose IVIG/PP + high dose IVIG/high dose IVIG alone	102	37.2	5	70.7	92.5

Table 5.

Graft and patient survival after HLAi transplantation.

4.2 Acute rejection

The risk of acute rejection is higher in HLAi renal transplants than in HLA-compatible, 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].

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.

5. Desensitization regimens

Desensitization aims to increase access to kidney transplantation in broadly sensitized individuals and to prevent ABMR after transplantation. In renal transplantation, desensitization could be done in a living or deceased donor setup. However, it is only performed in the deceased donor settings in heart and lung transplantation due to the nature of the donor and the organ transplanted.

In the living donor scenario, desensitization is planned well before surgery. In the deceased donor program, desensitization is attempted in the pre and peri-transplant period.

The desensitization protocols vary from center to center. Consensus is lacking on a standardized desensitization protocol among renal the transplant community. Most protocols use the following interventions in combinations (Table 6).

Mechanism	Intervention
Immunomodulation	Immunoglobulins (IVIG)
Antibody depletion	Plasmapheresis or adsorption
B cell depletion	Rituximab

Table 6.

Therapeutic agents for desensitization in renal transplantation.

5.1 Immunoglobulin (IVIG)

IVIG predominantly contains IgG (90-99%) from pooled human plasma. The exact mechanism of action of IVIG is unknown; however, it exerts immunomodulatory effects via multiple pathways. It blocks the FC receptors on plasma cells, thus preventing the rebound of DSA. Furthermore, blocking the FC neonatal receptor increases the lysosomal degradation of the circulating IgG [29]. Some researchers previously reported hemolysis with high-dose IVIG administration [30]. It was linked with the presence of anti-A/B isohemagglutinins [31]. It led to improvements in IVIG manufacturing, and the modern formulations carry a low risk of hemolysis [32].

High-dose IVIG (2 g/kg) alone enabled renal transplantation in 13/15 highly sensitized patients in a study [33]. In another study, 2 g/kg IVIG was administered before and after renal and heart transplantation in 45 CDCXM positive candidates [34]. Crossmatch became negative in 35 cases and reduced to positive FCXM in the remaining seven candidates. Forty-two candidates were transplanted eventually. Acute rejection occurred in 31% of cases, and 7% lost graft. At 24 months, 89% had a functioning graft. Investigators from Cedar Sinai Center reported successful renal transplantation in highly sensitized candidates using high-dose IVIG and induction therapy [35, 37, 38]. Graft outcomes varied according to immunologic risk. Graft survival was 94–96% and 40–100% at 1 and 2 years, respectively. Fifteen to 35% of cases experienced ABMR.

A randomized trial of 48 sensitized individuals (PRA > 50%) compared high-dose IVIG for 4 months to placebo [39]. IVIG shortened the time to transplantation from 10.3 to 4.8 years. Thirty-five percent of candidates in the intervention group received transplants compared to 17% in the control group. Rejections occurred in 53% of cases in the IVIG group while 10% in the control group. Graft survival was 80% in the IVIG group and 75% in the control group at 30 months.

5.2 Apheresis

Apheresis has been part of desensitization regimens since the 1990s [40]. Apheresis removes preformed antibodies from the plasma. Various apheresis modalities are used in desensitization though the ideal apheresis technique in desensitization is not known yet. Table 7 compares various apheresis modalities. A single-center, retrospective study looked at different apheresis modalities [41] in 45 DSA-positive renal transplant candidates. All modalities decreased DSA, but immunoadsorption (IA) and plasma exchange (PE) were more efficient than double filtration plasmapheresis (DFPP). DFPP is associated with severe adverse events, e.g., hypotension, high leukocyte counts, and low fibrinogen levels.

	Plasma exchange (PE)	Double filtration plasmapheresis (DFPP)	Immunoadsorption (IA)
Duration	2 hrs	3–4 hrs	6–8 hrs
Plasma volume processed	1–1.5	1–2	2–3
Substitution solution	+++	+	—
Removal of coagulation factors	++++	++	—
Selectivity for antibody removal	+	++	++++

Table 7.

Comparison of plasmapheresis techniques.

IA is frequently used in Europe, while PE is standard in the United States. Bleeding complications are more frequent with PE [42]. Giving time for fibrinogen to return to normal or repletion with cryoprecipitate before surgery reduces the risk of bleeding.

A retrospective case-control study compared desensitization with PE (1–12 sessions) + IVIG (100 mg/kg) in FCXM-positive renal transplant recipients to FCXM-negative controls [43]. Graft loss was high in the FCXM positive group at 9-year (14 vs. 7) [HR 2.6 (95% CI 1.03–6.4) t^1/2 = 6.8 yr.] Graft survival at 1 and 5 years were 89.9 and 69.4% in the FCXM positive group compared to 97.6 and 80.6% in the control group. In conclusion, the medium- and long-term outcomes of positive FCXM living kidney recipients are not promising. The results are comparable to extended criteria deceased donor renal transplantation.

A prospective study compared IVIG alone with two different combinations of PE, IVIG, and other interventions (Table 8) [44]. The author concluded that PE regimens result in lower ABMRs than IVIG-only regimens. Moreover, despite achieving a negative crossmatch, the rejection rate remained high in all the groups.

Pre-transplant desensitization regimen (n=)	Achieving negative cross match (%)	ABMR at 6 weeks (%)
High dose IVIG alone (n = 13)	36	80
PE + low dose IVIG + rituximab (n = 32)	84	37
PE + low dose IVIG + rituximab + pre-transplant ATG + DSA monitoring (n = 16)	88	29

Table 8.

Summary of study outcomes.

A pilot study analyzed the efficacy of two desensitization regimens in sensitized kidney transplant candidates who received deceased donor grafts [45]. The results (shown in Table 9) illustrate that intense immunosuppression on day 0 could improve long-term graft outcomes.

Desensitization regimen	Acute ABMR (%)	Microcirculation inflammation at 1 yr	Transplant glomerulopathy rate at 1 yr (%)	Chronic ABMR rate at 1 yr	eGFR at 1 year (ml/min/1.73 m^2)
IVIG (2 g/kg on day 0, 21, 42 and 63) [n = 36]	19.6	2.7 ± 0.2	38	41.1	43 ± 16
IVIG (2 g/kg) + PE (9.8 ± 4) + rituximab (375 mg/m^2 on day 4) [n = 18]	16.6	1.8 ± 0.2	7	13.3	54 ± 16

Table 9.

Study result summary.

Another retrospective case-control study (n = 48) examined five desensitization protocols [46]. The participants were stratified into five based on the MFI levels of the immunodominant DSA. Acute ABMR (25 vs. 12.5%) and T cell mediate rejection (TCMR) (23 vs. 14%) were high in desensitized recipients. However, graft loss and patient survival were similar across the groups.

5.3 Anti-CD-20 therapies

Rituximab is a chimeric anti-CD20 monoclonal antibody. FDA initially approved it for lymphoma; however, it plays a role in autoimmune disorders and SOT. A combination of rituximab (1 gm on day 7 and 22) and high-dose IVIG (on day 0 and 30) showed a significant reduction in PRA (44 vs. 77%) in Phase I/II trial (n = 20) [38]. Although the ABMR rate was high at 31%, graft survival was 94% at 1 year.

In another study, the investigators desensitized 76 highly sensitized patients with rituximab and IVIG combination [37]. PRA declined significantly from 79.7 ± 25.6% to 67.1 ± 28.6% for class I and 59.7 ± 29.2% to 49.7 ± 27.8% for class II anti-HLA antibodies. Fifty-nine percent of the participants received deceased donor kidney transplants. Graft survival was 84% at 24 months, but the ABMR rate was high at 37%.

A combination of rituximab and high-dose IVIG enabled renal transplantation in 71% of candidates in another study [47]. Transplantation with desensitization was more cost-effective than continuing dialysis.

A retrospective study compared desensitization with plasmapheresis and low-dose IVIG combination with or without rituximab (375 mg/m²) [48]. The rebound of DSA and non-DSA anti-HLA antibodies were lower with the rituximab group (7 and 33% respectively) compared to the no rituximab group (32 and 55% respectively) at 1-month post-transplantation. However, rituximab did not alter the ABMR rate, 5-year allograft survival, and elimination of HLA antibodies.

A randomized control trial compared high-dose IVIG + rituximab (1 g) with high-dose IVIG + placebo in sensitized renal transplant candidates [49]. Six patients in the intervention and seven in the placebo arm received deceased donor kidney transplants. DSA levels at transplantation were not different, but ABMR was seen only in the placebo arm. No graft loss occurred in the rituximab arm compared to 2 in the placebo arm. The study was prematurely stopped due to poor outcomes in the placebo arm.

Obinutuzumab is a type II anti-CD20 monoclonal antibody used in refractory and relapsing hematological malignancies. It is more efficient than rituximab in depleting B-cells in lymphoid organs. An open-label phase I trial (n = 25) assessed the efficacy of obinutuzumab + IVIG (2 g/kg) in sensitized renal transplant candidates [50]. MFI levels decreased significantly but were clinically irrelevant. Nine patients developed severe adverse events, mainly infectious complications. Though encouraging, these results need further validation.

In summary, the combination of apheresis + IVIG and or B cell-depleting agent is the most used desensitization protocol in renal transplantation.

The exact sequence and doses of the desensitization therapies vary among the centers. We perform low and intermediate risk living donor HLAi incompatible renal transplants at our center. We do not perform CDCXM positive transplants. Low risk HLAi renal transplant candidates (DSA positive but negative FCXM) are desensitized with 1 gram/ kg single IVIG dose. Patients with intermediate risk HLAi renal transplants [DSA positive and FCXM positive (≤300 median channel shift)] receive a single dose of rituximab 500 mg IV with pre-medications a week before transplantation. Two doses of IVIG 1 gram/kg/day (max dose 140 gm) are administered on subsequent days. PE or IA is not used in HLAi transplant desensitization at our center. We do not repeat FCXM after desensitization therapy.

In deceased donor kidney transplantation, due to the unpredictable timing of the organ availability, pre-transplant desensitization is offered only to those within 1 year of receiving renal grafts. High-dose IVIG and rituximab are given in the same protocol as the living donors. Luminex single antigen testing is performed monthly to maintain the current sera for crossmatch testing. A second course of desensitization, consisting of PE sessions followed by high-dose IVIG and rituximab, is offered if a patient does not receive a transplant within six months. PE (1.5 plasma volume with 5% albumin replacement) is performed on alternate days. IVIG 2 gram/kg (maximum dose of 140 g) is delivered immediately following the last plasmapheresis session. Rituximab 375 mg/m² is given one week after the last PE and IVIG session to avoid IVIG saturation of the Fc receptors [51]. This protocol is repeated every 6 months until the patient receives a transplant.

5.4 Alternative approaches

Bortezomib is a reversible proteasome inhibitor. It induces apoptosis of the differentiated plasma cells. A retrospective study of 44 sensitized patients (PRA > 20%) with positive CDCXM or a FCXM were desensitized with 1–2 cycles of bortezomib 1.3 mg/m² (6–8 doses) + rituximab (375 mg/m²) and plasmapheresis [52]. Nineteen patients received kidney grafts from living donors. Immunodominant DSA declined 51.1% in 83% of the cases. ABMR rate was 12.5%, and graft survival was 94.7% at 1.2 yr.

In another study, 32 doses of bortezomib 1.3 mg/m² alone were administered for desensitization in 10 highly sensitized renal transplant candidates. Although MFI values declined after bortezomib, crossmatch, and cPRA remained unchanged [53]. Two patients discontinued therapy due to side effects and two had to reduce the dose.

Carfilzomib is an irreversible proteasome inhibitor. It has a sustained and durable effect on plasma cells compared to bortezomib. Furthermore, it has better tolerability than bortezomib. A combination of 12 escalating doses of carflizomib (20–32 mg/m²) + PE (minimum three sessions) was trialed in 13 sensitized patients (mean PRA 97%) [54]. The immunodominant DSA declined by >50% in 70% of cases; however, the antibodies rebounded within 5 months after treatment discontinuation.

Eculizumab is a humanized monoclonal antibody against terminal complement component C5. It is used in ABMR treatment; however, experience in desensitization is limited. Thirty renal transplant candidates with initial B cell positive crossmatch (mean channel shift 200–450) were administered 1200 mg eculizumab immediately before transplantation, followed by 600 mg weekly dose for 4 weeks [55]. Patients with mean channel shift >300 were treated with plasmapheresis before transplantation to bring it down to <300. The outcomes were compared with a historical control who received the same desensitization protocol but without eculizumab. ABMR incidence was low in the eculizumab group, but the DSA rebounded.

A study compared the C1-inhibitor to placebo as a desensitization agent. Twenty highly sensitized patients (PRA > 50% and positive FCXM) who received plasmapheresis + rituximab + IVIG were randomized to C1-inhibitor or placebo [56]. ABMR was low in the intervention arm (20 vs. 30%, p = 0.6). Further validation is needed.

5.5 Investigational agents

Imlifidase is a recombinant IgG endopeptidase produced by Streptococcus pyogenes. It cleaves all sub-classes of IgG into Fc and F (ab) fragments. European Medicine Agency (EMA) has granted conditional approval for Imlifidase in desensitization, but the US Food and Drug Administration (FDA) is yet to approve it.

Two independent phase I/II studies (n = 25) were conducted in the United States and Sweden in highly sensitized patients (cPRA 96% and 81% respectively) [57]. Eight percent of participants had positive FCXM, and 92% had DSA. Imlifidase (0.24 mg/kg in the USA and 0.25 mg/kg or 0.5 mg/kg in Sweden) was given to all patients 4-6 hours pre-transplant. Ninety-two percent of the candidates received deceased donor kidney transplants. The United States cohort received a high dose IVIG, rituximab, and alemtuzumab at induction, while the Swedish population received horse ATG at induction but no IVIG and rituximab. IgG antibodies in the recipient serums were cleaved within 6 hours of the infusion and remained absent for at least 7 days. IgG recovered gradually but remained low at 28 days after the infusion. DSA rebounded in the Swedish cohort between 7 and 14 days. In the United States cohort, DSA rebound was low. One patient from the US cohort had hyperacute rejection attributable to IgM DSA. The ABMR rate was 27% in the Swedish cohort at two weeks, while only 14% experienced ABMR at 2 and 5 months in the US cohort. Mean GFR at 1–6 months was 70 and 49 ml/min/1.73 m² in the United States and Swedish cohorts, respectively.

Another multinational phase II study assessed the crossmatch conversion efficacy of Imlifidase in 19 highly sensitized kidney transplant candidates [58]. Eighty-nine percent (n = 17/19) of patients achieved a negative crossmatch within 24 hours of treatment. Thirteen patients received deceased donor kidney transplants, five received a living donor transplant, and one patient was not considered for transplantation due to a reaction to Imlifidase. DSA rebounded within 3–14 days after the treatment, though MFIs were lower than the pre-transplant level. Patient and graft survival at six months was 100% and 89%, respectively. Half of the patients experienced acute rejection; most (7/9) were ABMR.

A retrospective study examined 3 years of follow-up data of 39 crossmatch positive patients who received renal transplants [59]. Thirty-eight percent (n = 15/39) experienced ABMR. Overall graft survival was 84% at 3 years, notably lower in those who experienced ABMR (77 vs. 93%). Imlifidase has shown promising results, but further research is needed.

Clazakizumab- is a humanized, anti-interleukin (IL)-6 monoclonal antibody. It is used in chronic active ABMR in kidney transplantation, but experience as a desensitization agent is limited. Twenty highly sensitized patients (mean cPRA 96%) were desensitized with plasmapheresis, high-dose IVIG, and clazakizumab (25 mg * 6 doses) in a pilot study [60]. Eighteen patients received deceased donor renal transplants. Clazakizumab was continued for 12 months post-transplantation. MFI levels declined in almost all patients. Rejection occurred in four cases; one graft was lost due to a technical problem. Patient survival was 100 percent at the end of the study. Further studies are needed to validate these findings.

Belimumab is a human monoclonal antibody that inhibits B cell activating factor. However, it failed to show notable results in a pilot study as a desensitization agent [61]. Similarly, Atacicept, another monoclonal antibody targeting B cell activating factor, did not achieve the desired results in pre-clinical desensitization studies [62, 63].

Daratumumab (anti-CD38 antibody) and anti-C-X-C chemokine receptor type 4 decreased DSA in animal models and improved graft survival [64]. Validation in clinical trials is awaited.

5.6 Post renal transplant monitoring

Monitoring protocols vary from center to center. Some centers monitor HLAi renal transplant recipients like HLA compatible patients but keep a low threshold for renal biopsy in case of graft dysfunction. Other centers prefer protocol renal biopsies at pre-defined time points. Most centers monitor DSA after HLAi transplantation, though the frequency of monitoring varies. In our center, DSA are monitored monthly for the first 3 months, every 3 months up to 12 months, and then yearly. IVIG given during the pre-conditioning therapy could show false positive DSA due to non-specific binding of IVIG to SAB, but this effect fades away after 2 months. Renal biopsy is performed in patients with persistent de novo DSA to rule out ABMR.

6. Blood type incompletable (ABOi) renal transplantation

Blood type or ABO incompatibility (ABOi) poses another barrier to SOT. Earlier attempts to cross the ABO barrier without desensitization failed [65]. It was an absolute contraindication for transplantation due to the higher risk of ABMR and graft failure. However, the shortage of deceased organ availability (especially in Japan) fueled the re-exploration of this avenue. Moreover, approximately one-third of the living donors have ABOi with their potential recipients. ABOi renal transplants are performed globally thanks to desensitization. The outcomes of ABOi renal transplants are comparable to ABO-compatible transplants [66].

Blood group antigens are present on red blood cells, platelets, lymphocytes, endothelial cells, and epithelial cells. Human blood is classified into four blood groups, i.e., A, B, AB and O. Blood group antibodies called isohemagglutinins, are formed against non-self-blood group antigens. For example, an individual with blood group A will have antibodies against B antigens, while a person with blood group O will have antibodies to both A and B antigens.

Blood group O donors can donate to any blood group. In contrast, blood group AB recipients can receive grafts from all blood group types without desensitization. The blood type frequencies in the population vary; however, blood groups A and O are the most common. Renal transplant candidates with blood groups O and B wait longer on the waiting list for compatible donors. In the context of ABOi transplantation, both IgM and IgG isohemagglutinins are clinically relevant.

It is also important to note that individuals with the blood group O produce higher Isohemagglutinins levels than those with blood type A or B. Therefore, the risk of ABMR is higher with blood type O after ABOi transplantation.

Blood type A consists of two sub-groups: A1 and non-A1. A1 type occurs in 80% of the population, while non-A1 is observed in 20% of the US population. The non-A1 type carries low immunologic risk due to lower antigenic expression.

The measurement and reporting of ABO isohemagglutinin vary from center to center [67]. Isohemagglutinins titers are measured by the dilution method. Interpretation of this method is observer-dependent, and the outcomes are semi-quantitative. For example, the actual value of isohemagglutinins titer 1:8 could range between 1:4 and 1:32. Some centers use column agglutination technology, but it is expensive.

6.1 Desensitization in ABOi renal transplantation

The purpose of ABO desensitization is to reduce pre-transplant anti-A/B isohemagglutinins, reducing the risk of ABMR, and allowing successful transplantation. ABO desensitization therapy is commenced well before the transplant surgery. It is offered presently in the living donor set up only. However, sporadic cases of deceased donor ABOi renal transplants are reported [68, 69]. There is a lack of consensus about a standard ABOi desensitization protocol. Therefore, protocols are center specific. However, like HLAi transplantation, most centers use the following treatments in various combinations,

• Aphaeresis—The risk of acute ABMR is high in patients with higher pre-transplant isoagglutinin titers [70]. Apheresis reduces the circulating anti-A/B isohemagglutinins to a predetermined titer. Most protocols consider isohemagglutinins titers (IgG and IgM) ≤ 1:8 safe for transplantation. Some centers perform transplantation with isohemagglutinins titers up to 1:32 [71]. Most centers decline candidates for transplantation with baseline isohemagglutinins titers ≥1:256 for both IgM and IgG using the tube dilution method. One PE session (1.5 plasma volume) reduces isohemagglutinins titer by one dilution. The baseline isohemagglutinins titers help in calculating the dose and frequency of apheresis.

• IVIG- Most centers administer low-dose IVIG (100 mg/kg) after each apheresis to replace the eliminated immunoglobulins. Moreover, it also causes immunomodulation, as explained in section 4.1.

• Splenectomy and B cell depleting agent—Before the rituximab era, splenectomy was part of the ABOi desensitization protocol [72]. Nowadays, splenectomy is rarely practiced, thanks to the introduction of rituximab.

ABO desensitization therapy is commenced before the scheduled surgery date; however, the time and sequence of desensitization therapy varies according to the center.

In our center, a single rituximab dose (500 mg IV) is administered with pre-medications 2 weeks before surgery. PE is initiated 1 week before the operation. Each exchange is followed by a 100 mg/kg IVIG replacement. Three doses of ATG (1.5 mg/kg/day) are administered at induction. Patients are maintained on a triple maintenance regimen. A/B isohemagglutinins titers are checked daily for 2 weeks. After a fortnight, the allograft develops immunologic accommodation to low levels of A/B isohemagglutinins and resists complement-mediated damage. Additional PE and renal biopsy are considered with rising isohemagglutinins titers and serum creatinine levels within the first two weeks. Some units commence desensitization and maintenance immunosuppression simultaneously a month before the scheduled surgery. PE has not been considered in some centers if A/B isohemagglutinins titers are ≤1:16 [73], but higher rejection rates are reported with this practice.

There is a lack of high-quality evidence to guide optimal desensitization protocol for ABOi renal transplantation; however, non-randomized studies data show the efficacy of the combination therapies described above. Plasma exchange and immunoadsorption effectively remove isohemagglutinins [74]. Although IVIG is used in most ABOi desensitization protocols, this practice has not been trialed in randomized studies. Moreover, the optimal dose of IVIG is also not clearly defined. Rituximab and splenectomy are not directly studied. A systematic review showed similar graft and patient survival comparing splenectomy-based and rituximab-based protocols [75]. Some researchers have reported successful ABOi renal transplantation without using rituximab; however, they employed daily plasmapheresis for at least 2 weeks to keep isohemagglutinins titers ≤1:16 [76].

6.2 Outcomes and cost of ABOi renal transplantation

The risk of complications is high in the first few years after ABOi renal transplantation. A meta-analysis reported higher mortality in the first 3 years after an ABOi transplant vs. ABO compatible renal transplant [77]. However, this difference fails to exist at 8 years or more. Similarly, death-censored graft survival was low in ABOi vs. ABO compatible transplants, but this difference equalizes after 5 years. Furthermore, the risk of surgical complications, rejection, infections, and malignancy is higher in ABOi compared to ABO-compatible transplants.

The outcomes of ABOi and HLAi transplants are inferior to compatible transplants. However, desensitization is justified in selected patients due to long waiting time, high mortality on dialysis and low access to compatible donors.

ABOi renal transplantation bears a higher monetary cost. The average total cost of hospitalization was $65,080 for ABOi vs. $32,039 for compatible transplants [78]. However, the benefits of ABOi transplants outweigh its higher cost in the long run. Due to suboptimal outcomes, efforts should be made to avoid ABO incompatible transplantation; however, if it is not possible, ABOi transplantation with desensitization is an alternative.

7. Non-HLA antibodies

The most pertinent immunologic barriers in solid organ transplantation are HLA and ABO incompatibility however; rejection episodes are reported in the absence of DSA raising the possibility of non-HLA antibodies.

Several non-HLA antibodies against epithelial and endothelial proteins are associated with poor renal graft outcomes [79]. Angiotensin II type I receptor antibodies have attracted much attention [80]. Non-HLA antibodies are not part of the immunologic risk assessment currently however; they are worth consideration in some instances.

8. Outcomes of desensitization in cardiac and lung transplantation

Desensitization protocols in cardiac and lung transplantation are based on experience from renal transplantation. As in renal transplantation, variability in the desensitization protocols, clinical endpoints, and antibody measurement techniques also exist in cardiac and lung transplantation [81]. Most cardiac and lung desensitization studies are small, have shorter follow-up periods, and report inconsistent results. Protocols vary between centers, but most comprise apheresis, IVIG, and rituximab combinations. Desensitization in cardiac and lung transplantation is conducted in a deceased donor setup, considering the unique characteristics of the individual patient and the nature of the organ being transplanted. Some centers commence desensitization before transplantation, while others administer it in the perioperative period. Below is a brief account of a few pertinent studies.

In a case-control study, thirty-five sensitized (PRA > 10%) cardiac transplant candidates were desensitized with five sessions of PE (1.5 plasma volume) and 20-gram IVIG administered after every PE. All patients underwent cardiac transplantation. Seventeen had positive CDCXM before desensitization. Negative crossmatch was achieved in seven cases. Both class I and class II DSA declined significantly post-treatment. Survival was better than the control group; however, more rejections were observed in the intervention group [81].

In another study (n=16), sensitized cardiac transplant candidates (PRA >10%) were treated with a single session of PE (1.5 plasma volume with albumin + four units FFP replacement) and 20-gram IVIG followed by triple maintenance immunosuppression regimen [82]. PRA declined from 55.5 to 34.9%. Survival and rejection rates at 22 months were similar among the intervention and control groups.

In a large observational study, 21 sensitized (PRA > 10%) cardiac transplant candidates were desensitized with plasma exchange, 2 g/kg IVIG, and rituximab (375 mg/kg² in refractory cases) [83]. Mean PRA declined from 70.5 to 30.2% post-desensitization. Five-year survival was 71.4% in the desensitized group compared to 81.1% in the untreated sensitized and 75.7% in the unsensitized group. ABMR rates were higher in the desensitized group (66.7%, 89.2% and 96.5%, respectively). Vasculopathy rates were similar between the groups.

In a small study, 16 sensitized left ventricular assist device (LVAD) recipients were treated with IVIG and a single dose of cyclophosphamide. Class I HLA DSA declined by 33%, but the DSA reappeared at four weeks [84]. In another study, a weekly 500 mg /kg IVIG dose reduced PRA in three LVAD recipients to 23% at one month and 52% at two months [85].

Eighteen highly sensitized lung transplant candidates (PRA ≥ 80%) were desensitized with methylprednisolone, bortezomib, rituximab, IVIG, and PE [86]. Nine patients were transplanted; two were on the waiting list at the time of publication, while seven patients were removed from the waiting list due to medical reasons. This protocol failed to reduce pretransplant PRA.

Another retrospective study desensitized fifty-three lung transplant candidates in the perioperative period with PE, IVIG, and anti-thymocyte globulin [87]. The sensitized cohort had preformed DSA, but only five had positive CDCXM. Clinical outcomes were like the unsensitized cohort.

9. Conclusion

Highly sensitized patients face longer waiting times on the transplant list than unsensitized patients. They experience greater morbidity and mortality. Sensitized patients have limited access to compatible organs. There is an unmet medical need to expand their access to transplantation.

Efforts have been made to circumvent incompatible transplants in sensitized patients, e.g., utilizing potential living donors, increasing the chances of well-matched organs via priority points in the regular allocation, acceptable match program, and kidney-paired donation. Although successful to a certain extent, such measures have failed to provide compatible organs to all sensitized patients. Desensitization could facilitate access to transplantation in selected sensitized patients who fail to access compatible organs.

It is worth noting that the outcomes of desensitization are inferior to compatible transplants. However, compared to dialysis, incompatible transplantation is cost-effective, enhances quality of life, and confers survival benefit. Acute and chronic ABMR risk is high in incompatible transplantation and is a significant cause of allograft loss. Infections and malignancies are frequent due to higher cumulative doses of immunosuppressive drugs.

Implementing novel and innovative strategies to avoid crossing the immunological barriers is vital. Transplantation tolerance has the potential to minimize sensitization. HLA epitope matching is a step forward in lowering post-transplant sensitization. Newer biomarkers are needed for immune monitoring and early diagnosis of graft rejection. Running multiple desensitization randomized trials is not feasible due to limited resources and finite eligible patients. Innovative trial designs such as master protocols could offer testing multiple therapeutics in one trial, thus accelerating the development of effective protocols and therapies. Moreover, standardizing HLA antibody and cPRA measurements will be a significant achievement in this field.

Acknowledgments

This work is not funded.

Conflict of interest

“The authors declare no conflict of interest.”

Notes/thanks/other declarations

My parents (Muhammad Jamil and Ismat Begum), my wife, and my children supported me in completing this work. I would also acknowledge the support of my supervisors, Dr. Matthew Howes and Ida Ryland.