Comparison of Single Antigen Bead-Based, Cell-Based, and Solid Phase-Based Crossmatch Methods.

AIM: Antibody assessment during pretransplantation term is important to detect donor specific antibodies. These donor-specific antibodies are determined by various crossmatch methods (flow cytometric [FCXM], complement-dependent cytotoxic [CDCXM], and Luminex [LMXM]). Recently, single antigen bead (SAB) assays have been used for the assessment of hypersensitized patients. The aim of this study was to compare sensitivity and specificity of the 3 crossmatch methods in reference to the SAB method. METHOD: In this study, 69 hypersensitized patients with high class I and/or class II panel reactive antibodies were tested using the flow cytometric SAB method. Serum samples were cross-matched by 3 crossmatch methods with the cells of a volunteer healthy individual, and the results were evaluated according to HLA and cross-reactive epitope groups (CREGs). RESULTS: Sensitivity was found to be better with T FCXM (0.91) and class I LMXM (0.87). Specificity of peripheral blood lymphocyte CDCXM method (1.0) was found to be better than the other 2 methods (0.33 and 0.57, respectively). Sensitivity of class II LMXM (0.88) was found to be better than the others (0.42 for B CDCXM and 0.82 for B FCXM, respectively). The specificity of the B CDCXM, B FCXM, and class II LMXM was similar (0.44, 0.44, and 0.33, respectively). CREGs results were similar to HLA results. CONCLUSION: Although CDCXM has high specificity for the detection of anti-HLA antibodies, it has low sensitivity. To increase sensitivity, FCXM or LMXM methods may be used with the CDCXM test. These results will be beneficial for laboratories and clinicians during graft survival and patient health assessment.

Investigation of Anti-HLA Antibodies of Highly Sensitized Patients by Single Antigen Bead and C1q Tests.

PURPOSE: The single antigen bead (SAB) test contributes to conventional cellular and solid phase crossmatch tests in renal transplantation. However, the determination of anti-HLA antibodies of the patients may not reflect the pathologic features of these antibodies. Highly sensitized patients produce antibodies against a number of HLAs; therefore, their transplantation chance decreases. In this study, we aimed to evaluate SAB and C1q test results of highly sensitized patients. METHOD: In this study, 33 end-stage renal failure patients with >80% panel reactive antibody were included. Of the patients, 58% (n = 19) were female, and 42% (n = 14) were male. The mean age was 46.2 ± 12.4. All of the serum samples were inactivated by heat before use. SAB and C1q tests were performed according to the manufacturer's instructions. RESULTS: We obtained statistically significant results between the positive bead counts and raw mean fluorescence intensity (MFI) values of 2 tests (P < .01 for class I and II). There was a statistically significant difference between the 2 tests in terms HLA-A, -C, -DR, and -DP MFI values, whereas HLA-B and -DQ MFI values were similar for the 2 tests. CONCLUSION: The difference of raw MFI values between the 2 tests may be due to the fact that the C1q test detects only IgG1 and IgG3 antibodies, whereas the SAB test can detect all IgG subtypes. We considered that anti-HLA-B and -DQ antibodies have high complement-fixing features; these antibodies should be investigated selectively due to the similarity of anti-HLA-B and -DQ antibody MFI values in the 2 tests.

A Comparative Study: Flow Cytometry, Complement-dependent Cytotoxicity, and Luminex Methods.

AIM: The cell-based flow cytometric and bead-based Luminex crossmatch methods have been used alongside the standard complement-dependent cytotoxic crossmatch (CDCXM) test to detect donor specific anti-HLA antibodies. In this study, it was aimed to compare flow cytometric crossmatch (FCXM), CDCXM, and Luminex donor-specific crossmatch (LM-XM) tests for pre-transplant assessment of patients who applied to Tepecik Education and Research Hospital for kidney transplantation from related or deceased donors. METHOD: HLA tissue typing of 1120 patients were tested using the sequence specific oligonucleotide probe method with low resolution. FCXM and LM-XM were performed according to the manufacturer's instructions. The CDCXM test was performed according to the standard procedure. The results were analyzed using SPSS version 21.0 software (IBM, Armonk, NY, United States). P < .05 was accepted as statistically significant. RESULTS: FCXM, CDCXM, and LM-XM tests were performed on 58.2% (n = 652), 91% (n = 1019), and 55.4% (n = 620) of 1120 patients. There were statistically significant differences between FCXM/CDCXM, LM-XM/CDCXM, and FCXM/LM-XM (P < .0001), although there was also a moderate correlation between them (for class I, r = .599, r = .693, and r = .507; for class II, r = .546, r = .471, and r = .495, respectively). The results obtained according to donor type were compatible with the total study group. CONCLUSION: The utilization of FCXM and/or LM-XM tests together with the CDCXM method before kidney transplantations from related and/or deceased donor may facilitate the determination of target cells of donor-specific antibodies or their antibody class, which may increase the success of transplantation.

Effect of MDR1 polymorphisms on the blood concentrations of tacrolimus in Turkish renal transplant patients.

BACKGROUND: Tacrolimus, a calcineurin inhibitör, is prescribed to prevent allograft rejection in renal transplantation. Tacrolimus not only has a narrow therapeutic index, but also shows significant interindividual differences. The absorption and metabolism of this drug are affected by multidrug resistance (MDR) 1 gene polymorphisms that correlated with single-nucleotide polymorphisms (SNPs) affecting in vivo P-glycoprotein activity. This study investigated associations of MDR1 gene C3435T polymorphism with tacrolimus blood concentrations and dose requirements as well as acute rejection episodes among Turkish renal transplant patients. METHODS: One hundred living-donor transplant recipients and 150 healthy control subjects underwent C3435T genotyping using polymerase chain reaction-restriction fragment length polymorphism. Blood concentrations of tacrolimus were determined with the cloned enzyme donor immunoassay. RESULTS: The CC, CT, and TT genotype frequencies among patients were, respectively, 44.0%, 33.0%, and 23.0% versus 36.7%, 43.3%, and 20.0% among control subjects. There was no significant difference between (P = .061; P = .102; P = .211; respectively). The ratio of blood concentration to dose of tacrolimus for patients with mutant homozygous 3435 TT genotype was higher than that of wild-type 3435 CC genotype homozygous individuals. The doses for these patients were lower at 1, 3, and 12 months (P = .048; P = .03; P = .041, respectively). There were no significant differences between the groups regarding coprescription of drugs that affect tacrolimus concentrations, such as diltiazem. Acute rejection episodes were not associated with the CC vs CT or TT genotypes: odds ratio (OR), 0.517 (95% confidence interval [CI], 0.190-1.407; P = .192); OR 1.558 (95% CI, 0.587-4.136; P = .372); OR 1.346; (95% CI, 0.456-3.968; P = .590), respectively. CONCLUSIONS: Determination of MDR1 polymorphism may help to achieve target of tacrolimus blood concentrations.

Comparision of anti-HLA antibodies of kidney transplant candidates with chronic renal failure by two different methods: Flow-PRA and Luminex PRA.

OBJECTIVE: The aim of the study was to compare anti-HLA antibodies examined as panel-reactive antibody (PRA) in kidney transplant candidates with chronic renal failure (CRF) with the use of 2 methods: Flow-PRA and Luminex-PRA. METHODS: CRF patients displaying class I PRA (n = 34) and/or class II PRA (n = 41) were tested by the 2 different methods from April 2012 to September 2012, using antigen-coated beads. RESULTS: Eleven (32.3%) 34 patients tested for class I PRA were female and 23 (67.7%) male; 17 (41.5%) 41 patients tested for class II PRA were female and 24 (58.5%) male. Only 2 patients were preemptive, the others had been subjected to dialysis. The concordance ratio of class I PRA test results between Flow-PRA and Luminex PRA was 67.6%. Whereas 13 samples (38.2%) were positive by Flow-PRA, 22 (64.7%) were positive by Luminex-PRA. Two of the 3 patients not previously immunized were found to be positive only by Luminex PRA; 1 was noted to be positive only by Flow-PRA. Regarding class II PRA screening, the concordance between Flow-PRA and Luminex PRA was 70.7%. Whereas 14 (34.1%) samples were positive for Flow-PRA; 24 (58.5%) were positive for Luminex-PRA. The 2 patients not previously immunized were positive only in Luminex PRA. CONCLUSIONS: We speculated that the reason for the low concordance ratios was due to the use of sera that had been previously found to be indeterminate in PRA tests. We also speculated that the low concordance ratios were due to the coating procedure for the beads, which may cause changes in antigenic epitopes and decrease concordance between Flow-PRA and Luminex-PRA.

Comparison of complement-dependent cytotoxic and flow-cytometry crossmatch results before cadaveric kidney transplantation.

AIM: The presence of HLA donor-specific antibodies (DSA) before kidney transplantation decreases graft survival. In this study, we compared crossmatch results of kidney transplantation candidates, for cadaveric renal donation between March 10, 2012, and September 7, 2012. MATERIAL AND METHOD: The 47 kidney transplantation candidates tested for crossmatch included 10 for cadaveric donor organs. Two crossmatch methods were performed: complement-dependent cytotoxic crossmatch (CDCXM) and flow cytometry crossmatch (FCXM). Spleen cells were used as the source of lymphocytes for all crossmatch tests. RESULTS: The T and B cell ratios isolated from spleen were 38.8% and 34.8%, respectively. The concordance ratio of the two methods was 76.6% with 23.4% discordant results. Regarding the discordant results, 4.2% were positive CDCXM but negative FCXM; 191%, negative CDCXM but positive FCXM. All patients displaying positive crossmatches had a previous immunization history. As a result, we speculated that the positive CDCXM but negative FCXM results were due to the washing procedures in the FCXM disturbing antigen-antibody complexes. We suggest at least two different methods to be performed for crossmatch tests before kidney transplantation. CDCXM detects immunoglobulin G1 (IgG1) and IgG3, which are critical for rejection. FCXM is able to detect all IgG subgroups because of its high sensivity. As a result we suggest that both CDCXM and FCXM are preferrable strategies to detect DSAs.

Flow cytometric evaluation of pregnancy-induced anti-donor immunization.

BACKGROUND: Living donor kidneys from spouses and children (from offspring to parents) are currently considered to be important organ sources. However, pregnancy-induced alloimmunization may provoke acute rejection episodes after kidney transplantation. being flow cytometry cross-match (FCXM) we studied donor-specific antibodies (DSAs) in the sera of recipients planned for living kidney transplantation from their spouse or children. When the FCXM was positive, we confirmed the existence of anti-human leukocyte antigen (HLA) antibodies using flow cytometry panel-reactive antibody (flow-PRA). PATIENTS AND METHODS: Between March 2005 and November 2010, we tested 85 pretransplantation sera from renal transplant recipients for DSAs. The recipients included 37 wives (group I) and 48 husbands (group II). FCXM-positive sera were tested using a flow-PRA screening method using HLA class I and class II antigen-coated beads. The mean recipient age was 48.1 ± 9.8 (range, 28-69) years and the mean donor age was 45.1 ± 11.1 (range, 23-69 years). RESULTS: Among group I were 18 (48.6%) FCXM-positive cross-matches; for group II, 5 (10.4%) cases (P = .001). Sensitized patients were 37.9% FCXM-positive, whereas nonsensitized patients were 3.7% positive (P = .001). FCXM-positive patients were re-evaluated for anti-HLA antibodies using flow-PRA. Seventeen of 18 group I tests (94.4%) were FCXM-positive, whereas 3 of 5 (60%) were positive among group II. CONCLUSIONS: We concluded that flow cytometry-based cross-match and PRA techniques can be used to detect anti-HLA antibodies using spousal or children donors for kidney transplantation.

Flow cytometric crossmatching and panel-reactive antibodies in chronic renal failure patients.

AIM: Anti-donor antibodies, denoted as "panel-reactive antibodies" (PRAs), are one of the most important factors influencing graft survival after renal transplantation. PRA is generally analyzed with enzyme-linked immunosorbent assay or flow cytometry (FC), which identify the HLA antigen specific for the preformed antibody. PATIENTS AND METHODS: We tested 66 patients for FC crossmatch (FCXM) when they were called for cadaveric renal transplantation. Thirty of 66 patients were FCXM-positive; 36 were FCXM-negative. Among the FCXM positive crossmatches, 21 were T- and B-cell positive; seven B positive; and two T positive. The HLA antibodies in the sera of FCXM-positive patients were reanalyzed using flow-PRA. RESULTS: We detected HLA antibodies in 28/66 sera with flow PRA. The sera of 16/21 T-/B-, FCXM-positive patients contained both class I and II anti-HLA antibodies, five had only class I anti-HLA antibodies. One out of seven B-cell FCXM-positive patients had class I and class II anti-HLA antibodies, three, class I and 1 class II anti-HLA antibodies; the other two were negative. Class I and class II HLA antibodies were observed in two T-cell FCXM-positive patients. Four of 36 patients who were FCXM-negative were flow PRA positive: one had both class I and class II HLA antibodies and three, only class I HLA antibody. The comparison of FCXM and flow PRA results was significant (P = .001). CONCLUSION: FCXM results may be confirmed by flow PRA tests, an important method to differentiate HLA versus non-HLA antibodies.