Objectives: Crossmatch testing detects donor-specific anti-HLA antibodies to stratify immunological risk before living-donor kidney transplant, preventing antibody-mediated rejection. This study optimized a tiered strategy combining complement-dependent cytotoxicity, flow cytometry, and Luminex single antigen assays for precise pathogenicity assessment of donor-specific antibodies in resource-constrained Algerian centers lacking routine deceased-donor programs.
Materials and Methods: This prospective ongoing study (initiated in 2021) enrolled 90 sensitized patients (mean age 35 years; range, 9-61 years; sex ratio: 0.6) at Bab El Oued and Batna University Hospitals. Sensitizing events included transfusions (41%), pregnancy (13%), prior transplant (6%), and unknown (40%). Full 3-assay stratification was applied to 45 patients: category 1 (all positive tests) were excluded from transplant; category 2 (negative complement-dependent cytotoxicity crossmatch/positive flow cytometry crossmatch/positive Luminex single antigen assay) underwent desensitization to achieve negative flow cytometry crossmatch; category 3 (negative complement-dependent cytotoxicity crossmatch/negative flow cytometry crossmatch/positive Luminex single antigen assay) were subdivided into categories 3a (Luminex single antigen mean fluorescence intensity >3000, preventive desensitization) and 3b (mean fluorescence intensity <3000, direct transplant); and category 4 (all assays negative).
Results: Stratification was as follows: 1 patient in category 1, 4 patients in category 2 (1 transplanted after desensitization, others ongoing), 9 patients in category 3a, 24 patients in category 3b, and 7 patients in category 4. Of 31 transplanted patients, 90% reached 3-month and 60% reached 12-month follow-up, with mean serum creatinine of 1.4 mg/dL. Donor-specific antibody cleared in 80% (category 3b); 20% had persistent low mean fluorescence intensity donor-specific antibodies. No patients had acute antibody-mediated rejection on 12-month protocol biopsies.
Conclusions: Integrated crossmatch assays enabled safe transplant in 84% (categories 3b/4), reducing desensitization needs amid absent deceased-donor programs and rare paired exchange.

Key words : Antibody-mediated rejection, Donor-specific antibodies, Living kidney donor, Sensitization

Introduction
Kidney transplant provides superior quality of life and survival over dialysis for patients with end-stage chronic kidney disease.^1,2 Yet, preformed anti-HLA antibodies pose a major barrier, particularly donor-specific antibodies (DSA) that increase acute rejection risk.³ Crossmatch testing, a critical immunogenetic assay, detects DSA in recipient serum pretransplant to stratify risk and prevent antibody-mediated rejection. Two principal crossmatch approaches are employed: cell-based assays—including complement-dependent cytotoxicity crossmatch (CDCXM) and flow cytometry crossmatch (FCXM)—and solid-phase assays such as Luminex single antigen (LSA). Combining these methods enhances DSA detection, facilitating personalized risk assessment and informing living-donor eligibility, especially in Algeria, where deceased-donor and paired-donation options are limited Algerian sensitization rates of 50%, with 35.64% DSA-positive,⁶ exceed global rates ,^7,8 underscoring the need for refined strategies. Our prospective study evaluated this combined approach to enhance safety and access to transplant.

Materials and Methods
This ongoing prospective study, initiated in 2021, is assessing sensitized living-donor kidney transplant candidates at 2 Algerian centers: Bab El Oued and Batna University Hospitals. Some patients have undergone transplant; others remain in evaluation. Sensitization was evaluated via CDCXM, FCXM, and LSA (Luminex, One Lambda).^4,5 Classification followed European Guidelines (ENGAGE 2021)⁹ (Figure 110). Patients are classified into 4 categories based on CDCXM, FCXM, and LSA results. Category 1 includes patients with positive CDCXM, FCXM, and LSA, who are excluded from transplant. Category 2 comprises patients with negative CDCXM but positive FCXM and LSA, requiring desensitization to achieve negative FCXM. Category 3 involves negative CDCXM and FCXM but positive LSA, subdivided into category 3a (mean fluorescence intensity [MFI] >3000, necessitating desensitization to MFI <3000) and category 3b (MFI <3000, allowing direct transplant). Category 4 designates patients with negative results across all assays (despite prior LSA positivity), permitting direct transplant. Desensitization was performed using plasma exchange combined with intravenous immunoglobulin. For induction therapy, antithymocyte globulin was administered. Maintenance immunosuppression consisted of tacrolimus, mycophenolate mofetil, and corticosteroids. Antimicrobial prophylaxis included valganciclovir and cotrimoxazole for 6 months. Posttransplant monitoring involved DSA assessment at follow-up visits and protocol biopsies at 12 months.

Results
The study has enrolled 90 sensitized patients (sex ratio 0.6; mean age 35 years; range, 9-61 years). Sensitizing events included blood transfusion (41%), pregnancy (13%), prior transplant (6%), and unknown (40%). Full 3-technique stratification (CDCXM, FCXM, and LSA) was performed for 45 patients. Category 1 comprised 1 patient (2%) who was excluded from transplant. Category 2 included 4 patients (9%), of whom 1 underwent successful transplant following desensitization, whereas the others remain having ongoing evaluation. Category 3 included 33 patients (73%), with 9 patients in category 3a (MFI >3000) and 24 patients in category 3b (MFI <3000). Category 4 included 7 patients (16%). Thirty-one patients underwent transplant, with 90% available for 3-month follow-up and 60% reaching 12-month follow-up. At 12 months, the mean serum creatinine level was 1.4 mg/dL. Donor-specific antibodies cleared in 80% of patients (category 3b), whereas 20% exhibited persistent low-MFI DSA (<3000). None of the 12-month protocol biopsies revealed acute antibody-mediated rejection.

Discussion
This study showed the transformative potential of combined CDCXM, FCXM, and LSA crossmatch techniques in Algeria’s resource-constrained transplant landscape, where sensitization burdens 50% of candidates, a rate far surpassing French (Biomedicine Agency) and UNOS rates of approximately 30%.^7,8 In our centers, transplant is contraindicated if the CDCXM is positive (category 1). Category 2 cases, characterized by a positive FCXM, undergo desensitization protocols to achieve a negative FCXM before transplant. Category 3 cases are initially defined by a negative FCXM; however, our centers preferentially implement preventive desensitization when LSA MFI is >3000 (category 3a), targeting an MFI of <3000 to enable safe transplantation. Outcomes affirmed efficacy: among 31 transplants, none had subclinical antibody-mediated rejection at 12 months, creatine level was stable (1.4 mg/dL), and DSA clearance was shown in 80% in category 3b. Desensitization proved successful in 5 of 14 patients (categories 2 and 3a), thereby mitigating extended dialysis dependence in a setting characterized by only episodic deceased-donor transplants and a single instance of paired kidney exchange. Sensitization events reflect global epidemiological patterns, with transfusions predominant (41%); our young cohort (mean age of 35 years) accentuates the urgency for intervention in a high-morbidity, dialysis-dependent population. Strengths of our study include prospective design across 2 centers, multimodal DSA monitoring, and protocol biopsies, yielding robust short-term data. This approach curtails unnecessary interventions: avoiding desensitization in category 3b and 4 slashes costs and infection risks. Compared with reliance on a single assay, our combination of 3 boosts specificity, with 34% of sensitized patients receiving transplants. Our study has some limitations, including ongoing recruitment capping long-term survival analysis (median follow-up of ~6 months) and small group sizes in category 2 and 3a (n = 13), limiting power analyses. In addition, geographic centralization (Algiers and Batna) may not generalize results nationwide. Future directions include extending investigations to multicenter trials and economic modeling to quantify savings for dialysis aversion.

Conclusions
The Algerian model validates combined crossmatch protocols, integrating CDCXM, FCXM, and LSA, as a scalable, equitable innovation in histocompatibility testing that enhances identification of sensitized candidates requiring desensitization versus those suitable for direct transplant. By expanding eligibility without undermining immunological safety, rejection risk, infectious complications, or costs, our approach improves kidney transplant outcomes and provides a replicable framework for international programs lacking paired or deceased donor options, particularly in addressing HLA mismatch barriers.