The clinical significance of flow cytometry crossmatching in heart transplantation.

OBJECTIVE: Flow cytometry crossmatching (FCXM) is more sensitive than the cytotoxic crossmatch in identifying preformed antibodies to donor alloantigens, but its clinical importance is controversial. The objective of this study was to determine the association of a FCXM with survival and incidence of vascular rejection in cardiac transplant recipients with a negative cytotoxic crossmatch. METHODS: Between 1993 and 1998, 357 heart transplant recipients with a negative T cell cytotoxic crossmatch were studied by three-color FCXM to quantitate anti-donor IgG reactions against B and T lymphocytes. Reactions positive against both were consistent with human leukocyte antigen (HLA) Class I reactivity, and those against B cells only were considered to be against HLA Class II antigens. Endpoints were episodes of vascular rejection, death from acute and chronic rejection and overall survival. RESULTS: Fifty patients were FCXM for Class I-positive, 144 for Class II-positive, and 163 were negative. At 1 month, freedom from vascular rejection was 64% in Class I patients, but 90% and 96% in Class II or negative crossmatch patients (P<0.0001). Survival of the negative crossmatch group was higher than either Class I or II groups (94%, 74% and 76%, respectively, at 3 years; P<0.0001). Death from acute rejection was 3% and 2% at 3 years in negative or Class II-positive patients, but 19% in Class I patients (P<0.0001). Death from chronic rejection occurred only in Class II patients (P=0.002). CONCLUSIONS: Despite a negative T-cell cytotoxic crossmatch, a positive flow cytometry crossmatch correlates with important clinical events after heart transplantation.     

Platelet crossmatching for kidney transplants by flow cytometry

Although flow cytometry crossmatching is now performed by many kidney transplant centers, it yields a high false-positive rate, as evidenced in the present series by a 71% one-month success rate despite a positive crossmatch. In an attempt to reduce false-positive reactions while retaining the sensitivity of the flow cytometer, platelets were tested as targets. Whereas flow cytometry with T cells was not correlated with one-month failure rates, retrospective platelet crossmatching by flow cytometry showed that kidney failure within one month occurred in 53% of 19 patients with a positive platelet crossmatch compared with 15% of 48 patients with a negative platelet crossmatch. (P less than 0.002). Statistical significance in one-month outcome was also obtained when only first transplant patients were analyzed (P less than 0.04). The present results suggest that crossmatching with platelet targets may be a simpler way to avoid early primary nonfunction while minimizing false-positive reactions.     

Low Expression Loci and the Use of Pronase in Flow Cytometry Crossmatch

Pronase treatment of lymphocytes has been used to improve the specificity and sensitivity of flow cytometric crossmatch, especially B-cell flow cytometric crossmatch, due to the presence of Fc receptors on the cell surface. Some limitations have been reported in the literature: false negatives due to the reduction of major histocompatibility complex expression and false-positive T cells in HIV+ patients due to exposure to cryptic epitopes. This study aimed to evaluate the effect of pronase in our assays, using untreated and treated cells with 2.35 U/mL of pronase to improve flow cytometric crossmatch specificity and sensitivity. The study was carried out with donor-specific IgG antibodies (DSAs) to low expression loci (HLA-C, -DQ, or -DP) because, in our laboratory practice, patients with virtual crossmatch (LABScreen single antigen assays) to DSA against antigen HLA-A, B, and DR are excluded from cellular crossmatch. Our results showed that, for T-cell flow cytometry crossmatch (FCXM), a cutoff value of 1171 median fluorescence intensity (MFI), an area under the curve (AUC) of 0.926 (P < .0001), and 0.834 (P < .0001), a sensitivity of 100% and 85.7%, and a specificity of 77.5% and 74.4%, without and with pronase treatment, respectively. For B-cell FCXM without pronase treatment, the best cutoff was 2766 MFI, an AUC of 0.731 (P < .0001), a sensitivity of 69.6%, and a specificity of 66.7%, whereas for B cells treated with pronase, the cutoff value was 4496 MFI, an AUC of 0.852 (P < .0001), a sensitivity of 86.4%, and a specificity of 77.8%. Our analysis of 128 FCXM showed a better performance using the untreated lymphocytes for FCXM with the prerequisite of a higher cutoff value (≈5000 MFI) to reach a better sensitivity and specificity due to the loss of HLA expression.     

New crossmatch technique eliminates interference by humanized and chimeric monoclonal antibodies

Humanized and chimeric antilymphocyte antibodies (Ab) are used to prevent and treat rejection and for treatment of human disease. Rituximab (RIT, anti-CD20), daclizumab (DAC; anti-CD25), alemtuzumab (ALE; anti-CD52), or infliximab (IFX) may interfere with Ab detection methods such as complement-dependent cytotoxicity (CDC) and flow cytometric crossmatch (FCXM). These agents are recognized as anti-human Ab or fix complement and are not differentiated from anti-allo-Ab. A new enzyme-linked immunosorbent assay crossmatch (XM) utilizing class I and II HLA antigens from donor cells called Transplant Monitoring System (TMS; GTI, Waukesha, Wisc) potentially precludes interference by eliminating non-major histocompatability complex antigens. To test this, normal sera (nonsensitized volunteers) were supplemented with 0.1 or 10 microg/mL of RIT, DAC, IFX or ALE, and were tested using three methods: the TMS T-cell CDCXM with antihuman globulin (AHG); and B-cell CDCXM without AHG; and FCXM with mean channel shifts of 45 and 150 indicating positive T-cell and B-cell crossmatch, respectively. No reactivity occurred with normal sera using any crossmatch technique. At 0.1 and 10 microg/mL, RIT interfered with CDC B-cell, but not T-cell crossmatch. RIT at 10, but not 0.1 microg/mL interfered with B-cell FCXM. No interference occurred with RIT in T-cell FCXM or TMS. ALE interfered with B-cell and T-cell CDC and FCXM but neither class I nor II TMS. DAC did not interfere with CDC or FCXM at 0.1 microg/mL, but gave false positive B-cell FCXM and CDCXM with some samples. No interference by DAC occurred using TMS. TMS may be useful to differentiate de novo donor-specific Ab after treatment with humanized or chimeric Ab.     

Hyperacute and acute kidney graft rejection due to antibodies against B cells.

Because of the perception of its uncertain clinical significance, the B cell crossmatch is not universally performed before renal transplantation. Even though sporadic cases of hyperacute rejection associated with B cell antibodies have been reported, doubts remain in light of other studies suggesting no effect on graft survival. This report describes 4 cases of graft rejection (3 hyperacute and 1 acute) that occurred in patients with anti-B-cell antibodies specific against donor HLA-DR or DQ antigens. Absence of anti-donor class I antibodies was confirmed in all cases by 2-color flow cytometry. Strong evidence for an antibody-mediated mechanism was found in one patient with anti-class I and anti-class II antibodies in serum transplanted with a class II mismatched kidney. In this case, only anti-class II antibodies were recovered in the eluate of the nephrectomy specimen. These four cases were compiled from three different institutions over a four-year period, which confirms the infrequent occurrence of these events. While anti-class II antibodies may not always be detrimental for graft survival, these results also confirm that they have the potential to cause hyperacute or acute graft loss. We conclude that the information provided by the B cell crossmatch should be available at the time that a decision to proceed with a renal transplant is made.     

Pooled Human Gammaglobulin Modulates Surface Molecule Expression and Induces Apoptosis in Human B Cells

We have previously shown that the pooled human gammaglobulin (IVIG) inhibited mixed lymphocyte reaction (MLR). In this study, we examined (1) if IVIG contains blocking antibodies reactive with cell surface molecules required for alloantigen recognition and (2) if IVIG modulates these surface molecule expressions using flow cytometry. IVIG does not contain significant amounts of blocking antibodies against CD3, CD4, CD8, CD20, CD14, CD40, MHC class I and class II. It reduces the number of intact B cells and monocytes, reduces or modulates CD19, CD20 and CD40 expression on B cells, and induces morphological changes in B cells. This B-cell modulation results primarily because of apoptosis. IVIG also induces apoptosis in T cells and monocytes, but to a lesser degree. Induction of apoptosis requires the intact IgG molecule. Reduction of intact B cell and monocyte cell numbers, modulation of surface molecule expression on B cells, and deletion of B and T cells by apoptosis could result in inhibition of optimal T-cell activation. This likely represents the primary mechanism responsible for IVIG suppression of the MLR, and may account for many of the observed beneficial effects of IVIG seen in the treatment of human autoimmune and alloimmune disorders.     

Functional assessment of immune markers of graft rejection: a comprehensive study in live-related donor renal transplantation

A better understanding of the immunobiological processes and predictors of graft rejection holds promise for the development of potential therapeutic strategies and also individualization of immunosuppression. The objective of this study is to analyze the clinical relevance of immune parameters such as antidonor antihuman leukocyte antigen (anti-HLA) antibodies, monitoring of cytokines and their receptors on the graft outcome following live-related donor renal transplantation. Flow cytometry-based methods were used to detect antidonor antibodies (flow cytometry crossmatch, FCXM) and intracellular cytokines. Enzyme-linked immunosorbent assay (ELISA) methods were employed to detect anti-HLA class I and class II antibodies and quantitative serum-soluble interleukin-2 receptor (sIL-2R) levels. The data revealed that patients with HLA class I-specific IgG antibody experienced higher acute rejection (AR) episodes at 1 yr in comparison to the antibody negative group (82% vs. 56%, p = 0.01). On the contrary, donor-specific class II antibodies (B+) did not have any influence on the graft survival. However, 15 recipients having both T- and B-cell antidonor antibodies (T+B+) had significantly poor graft survival (60%) as compared to the antibody-negative group (T-B-, 82%, p = 0.05). Additionally, patients having non-donor but HLA-specific antibodies (FCXM-/ELISA+) had poor graft survival as compared to the antibody-negative group (64% vs. 88%, p < 0.05). Further, patients undergoing AR episodes had significantly higher expression of IFN-gamma-producing T cells (19.16 +/- 7.4% median 17.50) as compared to their pre-transplant levels (5.68 +/- 1.63%, Median 5.20) and the non-rejecter group (5.97 +/- 4.39%, median 4.3, p = 0.0004). Similarly sIL-2 was significantly increased in AR episodes during the first month of transplantation (292 +/- 131.5 pmol/L) as compared to those with well-functioning grafts (p = 0.01) and healthy controls (p = 0.001). Evaluation of antidonor antibodies by flow cytometry is found to be relatively more sensitive and a better predictor of graft outcome. Further monitoring of cytokine expression profile of primed peripheral T-helper cells and quantitative analysis of sIL-2R offer additional valuable diagnostic and prognostic tools for follow-up of transplant subjects and a better alternative for functional assessment of immunosuppression.     

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.     

Weak humoral posttransplant alloresponse after a well-HLA-matched cadaveric kidney transplantation

BACKGROUND: Screening of donor-specific antibodies or alloantibodies after kidney transplantation has not been performed routinely. The aim of this study was to evaluate the humoral antidonor and alloresponse of immunologically low-risk recipients of cadaveric renal allografts during the first posttransplant year. METHODS: Alloresponse against the donor was analyzed by means of T-cell immunoglobulin (Ig)G and IgM and B cell IgG flow cytometric crossmatch (FCXM) tests with sera from days 0, 21, 90, and 365 posttransplant. In addition, panel reactive anti-human leukocyte antigen (HLA) class I and class II antibodies (PRA I and PRA II) were analyzed using flow cytometric methods. The recipients were treated either with a low initial cyclosporine regimen with single-bolus antithymocyte globulin (ATG) or basiliximab induction or conventional cyclosporine triple therapy. RESULTS: No significant posttransplant anti-HLA class I or class II sensitization was found in the recipients as a whole. Recipients receiving a single-bolus ATG showed significantly higher proportion of PRA I positivity in the day 21 sample compared with the other groups. Flow cytometric donor-specific T- and B-cell IgG alloresponses remained low, but the proportion of T-cell IgM crossmatch-positive recipients increased during the study. Positive T-cell IgM FCXM was found to be associated with acute rejection episodes and cytomegalovirus (CMV) infections. CONCLUSIONS: In immunologically low-risk kidney-graft recipients, positive T-cell IgM FCXM at transplantation was found to be a risk factor for rejection episodes. Conversion of T-cell IgM FCXM to positive was found to be associated with CMV infections.     

Improved flow cytometric detection of HLA alloantibodies using pronase: potential implications in renal transplantation

BACKGROUND: Flow cytomeric crossmatch (FCXM) has grown in popularity and has become the "standard of practice" in many programs. Although FCXM is the most sensitive method for detecting alloantibody, the B cell FCXM has been problematic. Difficulties with the B cell FCXMs have been centered around high nonspecific fluorescence background owing to Fc-receptors present on the B cells and autoantibodies. To improve the specificity and sensitivity of the B cell FCXM, we utilized the proteolytic enzyme pronase to remove Fc receptors from lymphocytes before their use in FCXM. METHODS: Lymphocytes isolated from peripheral blood, spleen, or lymph nodes were treated with pronase and then used in a three-color FCXM. A total of 167 T- and B cell FCXMs using pronase-treated and untreated cells were performed. Testing used serial dilutions of HLA allosera (22 class I and 6 class II), with the titer of each antibody at one dilution past the titer at which the complement-mediated cytotoxicity anti-human globulin crossmatch became negative. RESULTS: After pronase treatment, the actual channel values of the negative control in both T cell and B cell FCXMs declined from 78+/-10 to 57+/-4 (P<0.05) and 107+/-11 to 49+/-3 (P<0.00001), respectively. Pronase treatment resulted in improved sensitivity of the T and B cell FCXM in detecting class I antibody by 20% and 80%, respectively. In no instance was a false-positive reaction observed. In this study, pronase treatment improved the specificity of B cell FCXM for detecting class II antibodies from 75% to 100% (P=0.03). In no instance was a false-negative reaction recorded. Lastly, on the basis of these observations we re-evaluated three primary transplant recipients who lost their allografts because of accelerated rejection. One of the patients was transplanted across negative T and B cell FCXM, whereas the other two patients were transplanted across a positive T cell, but negative B cell, FCXM. After pronase treatment, T and B cell FCXMs of each patient became strongly positive, and donor-specific anti-HLA class I antibody was identi. fied in each case. CONCLUSION: Utilization of pronase-treated lymphocytes improves both the sensitivity and specificity of the FCXM.     

Pretransplant HLA Antibodies Are Associated with Reduced Graft Survival After Clinical Islet Transplantation

Despite significant improvements in islet transplantation, long-term graft function is still not optimal. It is likely that both immune and nonimmune factors are involved in the deterioration of islet function over time. Historically, the pretransplant T-cell crossmatch and antibody screening were done by anti-human globulin--complement-dependent cytotoxicity (AHG-CDC). Class II antibodies were not evaluated. In 2003, we introduced solid-phase antibody screening using flow-based beads and flow crossmatching. We were interested to know whether pretransplant human leukocyte antigen (HLA) antibodies or a positive flow crossmatch impacted islet function post-transplant. A total of 152 islet transplants was performed in 81 patients. Islet function was determined by a positive C-peptide. Results were analyzed by procedure. Class I and class II panel reactive antibody (PRA) > 15% and donor-specific antibodies (DSA) were associated with a reduced C-peptide survival (p<0.0001 and p<0.0001, respectively). A positive T- and or B-cell crossmatch alone was not. Pretransplant HLA antibodies detectable by flow beads are associated with reduced graft survival. This suggests that the sirolimus and low-dose tacrolimus-based immunosuppression may not control the alloimmune response in this presensitized population and individuals with a PRA > 15% may require more aggressive inductive and maintenance immunosuppression, or represent a group that may not benefit from islet transplantation.     

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

AimAntibody 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.MethodIn 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).ResultsSensitivity 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.ConclusionAlthough 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.     

Novel Flow Cytometry-Based Method for Detection of Anti-HLA Complement Activating Donor-Specific Antibodies in Renal Transplant Recipients and Its Comparison With Other Conventional Detection Methods

BackgroundPresence of preformed donor specific antibodies (DSAs) detected by complement-dependent cytotoxicity (CDC-XM) is a strong contraindication for transplant. However, it has limitations including its sensitivity and its inability to distinguish between HLA-specific and other non-HLA-specific antibodies. In this study, we standardized CDC-XM by flow cytometry and determined its relevance by comparing its results with other methods of DSA detection, such as routine CDC-XM, antibody binding assay by flow cytometry (FC-XM), and Luminex-based crossmatch assays, such as Luminex crossmatch (LXM) and virtual crossmatch (VXM).Materials and MethodsA total of 79 serum samples were tested for DSAs by the flow cytometric complement-dependent cytotoxicity crossmatch assay (FC-CDC-XM) and then the results of FC-CDC-XM were compared with other detection methods such as CDC-XM, FC-XM, LXM, and VXM.ResultsWe found that the FC-CDC-XM assay is more sensitive than routine CDC-XM. Out of total 79 sera, 24 sera were detected positive (T cells positive: 1 case and B cells positive: 23) by FC-CDC-XM as compared with 3 sera using CDC-XM; these 3 sera also showed positivity by FC-CDC-XM. After FC-XM assay, 23 samples were positive by FC-XM and out of these 23 samples, 13 were also positive by FC-CDC-XM. On comparing the FC-CDC-XM results with VXM and LXM, 10 sera of 24 FC-CDC-XM positive had HLA class II antibodies detected on a Luminex platform.ConclusionsThe FC-CDC-XM is a more sensitive and specific method for detection of HLA-specific complement-fixing antibodies than CDC-XM and FC-XM. FC-CDC-XM should be used in tissue-typing laboratories after intra- and inter- laboratory validation.     

Donor-Specific Antibodies Adversely Affect Kidney Allograft Outcomes

The effect of low titers of donor-specific antibodies (DSAs) detected only by sensitive solid-phase assays (SPAs) on renal transplant outcomes is unclear. We report the results of a systematic review and meta-analysis of rejection rates and graft outcomes for renal transplant recipients with such preformed DSAs, defined by positive results on SPA but negative complement-dependent cytotoxicity and flow cytometry crossmatch results. Our search identified seven retrospective cohort studies comprising a total of 1119 patients, including 145 with isolated DSA-SPA. Together, these studies suggest that the presence of DSA-SPA, despite a negative flow cytometry crossmatch result, nearly doubles the risk for antibody-mediated rejection (relative risk [RR], 1.98; 95% confidence interval [CI], 1.36–2.89; P<0.001) and increases the risk for graft failure by 76% (RR, 1.76; 95% CI, 1.13–2.74; P=0.01). These results suggest that donor selection should consider the presence of antibodies in the recipient, identified by the SPA, even in the presence of a negative flow cytometry crossmatch result.In 1969, Patel and Terasaki published their landmark study that definitively established the detrimental effect of preformed donor-specific antibodies (DSAs) detected by complement-dependent cytotoxicity (CDC) crossmatch on short-term allograft survival, but they noted the limited sensitivity of the technique in the detection of preformed DSAs.¹ Since that time, technological advances have led to increasingly more sensitive tests for the detection of anti-HLA DSAs, including flow cytometry crossmatch and the more recent solid-phase assays (SPAs), such as Luminex.^2,3 The clinical significance of DSAs detected by sensitive SPA remains unclear, with disparate findings being reported in the literature.⁴ Further, the clinical utility of these tests, particularly for prognostication or modifying immunosuppressive therapy, when used individually or in some sequential manner remains unclear. Our aim was to perform a systematic review and meta-analysis of published reports of rejection rates and graft outcomes among renal transplant recipients with preformed DSA at levels detectable only by SPA but not by CDC or flow crossmatch.     

Identification of the antibodies involved in B-cell crossmatch positivity in renal transplantation

BACKGROUND: The significance of a positive B-cell crossmatch (BCM) in kidney transplantation has always been controversial in the evaluation of its implications on graft survival and specificity of the antibodies involved. METHODS: We have investigated the sera of 62 recipients of a kidney allograft transplanted across a positive BCM (T negative) for the presence of autoantibodies and anti-human leukocyte antigen (HLA) class I and II antibodies, using a combination of lymphocytotoxicity, enzyme-linked immunosorbent assay (ELISA), and flow cytometry tests. The controls were the 930 patients transplanted over the same period of time with a negative T and BCM. RESULTS: Autoantibodies were detected in 16%, and donor specific anti-HLA class II antibodies, mainly DQ, in 23% of the patients. None had antibodies against donor HLA class I. The target of the antibodies was not identified in 61%. Graft survival was comparable in the controls and in the +BCM patients, with nondonor-specific HLA reactivity. Patients with donor-specific anti-HLA class II antibodies had lower early graft survival and a higher incidence of vascular rejection. However, long-term allograft survival was similar to that of the other groups. CONCLUSION: These data suggest that in 77% of the patients, BCM positivity was not related with anti-HLA antibodies, and, in this case, graft survival was similar to that of the -BCM controls. In a minority of patients, anti-HLA class II antibodies were responsible for the +BCM, and their presence was associated with lower early, but not long-term, graft survival. Consequently, a +BCM should not systematically contraindicate kidney transplantation.     

A retrospective flow cytometric crossmatch study in transplant recipients with autoreactive lymphocytotoxic antibodies

Abstract Our previous data shows renal transplant recipients with autoreactive lymphocytotoxic antibodies to have a reduced transplant survival when compared to patients without autoantibodies. This could have been due to the presence of weak IgG antibodies inhibited by the dithiothreitol used to remove IgM antibodies in the pretransplant cytotoxicity cross-match. That possibility was investigated in a retrospective study of 52 recipients of 57 renal transplants who were recrossmatched using a more sensitive flow cytometry crossmatch (FCXM) to detect recipient IgG antibodies to donor T and/or B cell splenic lymphocytes. Fourteen of the 57 (24%) transplants failed. Six losses were within the 1st month posttransplant and four of these were immunological failures. None of the transplant failures had a positive pretransplant FCXM. These results showed that the recipients with autoantibodies did not have pretransplant IgG anti-donor antibodies. The transplant failures did not, therefore, relate to the presence of antibodies undetected by the dithiothreitol-treated cytotoxicity crossmatch.     

Outcome of kidney transplants in patients known to be flow cytometry crossmatch positive

BACKGROUND: The clinical significance of the flow cytometry crossmatch has been addressed in several retrospective studies, but the results have been controversial. There are no prospective studies in which patients known to be antibody positive underwent transplantation. METHODS: The flow cytometry crossmatch was performed prospectively in 1130 renal transplant recipients. A decision to perform transplantation was based on whether the positive results were on T or B cells, in the current or peak specimen, and taking into account the presence or absence of other immunological risk factors. One hundred antibody-positive patients received a transplant. Graft survival and rejection episodes were analyzed in this group and compared with 100 crossmatch-negative patients matched for age, sex, race, and time of transplantation. RESULTS: The incidence of rejection at 1 month was higher in antibody-positive patients (26%) than in antibody-negative patients (12%, P<0.01). Early rejection seemed to be more frequent in antibody-positive patients regardless of whether the antibodies were current or historic, or against T or B cells. There were more steroid-resistant rejections in antibody-positive than in antibody-negative patients. However, biopsy specimens showed that vascular lesions that can be associated with humoral rejection were not more frequent in the antibody-positive patients than in the controls. There were no differences in graft survival between the two groups. CONCLUSIONS: Low-level preformed alloantibodies detected by flow cytometry represent a risk of rejection even for patients purposely selected for having no additional immunological risk factors. The risk seems to be due to donor-specific memory rather than to a direct effect of the antibodies. The results indicate that flow cytometry provides useful information to assess donor-recipient compatibility.     

Renal graft survival is not influenced by a positive flow B-cell crossmatch

INTRODUCTION: The influence of a positive B-cell crossmatch on graft outcome in renal transplantation is controversial. METHODS: We analyzed graft survival using Kaplan-Meier estimates for recipients of deceased donor kidneys who were either regraft transplant patients (n = 198) from 1990 to August 20, 2004, or primary transplant patients (n = 361) from December 15, 2000 to August 8, 2004, each of whom had a flow T- and B-cell IgG crossmatch performed before transplantation. The flow B-cell crossmatch (FBXM) was not used to decide whether or not to transplant. Graft survival was analyzed by whether the patient's FBXM was positive or negative. We also evaluated creatinine levels and graft survival of 131 transplant patients (June 1, 2004 to July 1, 2005) by their FBXM result and by their HLA class II flow-defined IgG PRA. RESULTS: One- and three-yr graft survival for the primary transplant patient group with a positive FBXM (98% and 84%) was not significantly different from the group with a negative FBXM (96% and 93%) (log-rank = 0.9). Similarly, graft survival at one, five, and 10 yr for the regraft transplant group whose FBXM was positive (91%, 76%, and 61%) was not significantly different from the group whose FBXM was negative (91%, 79%, and 77%) (log-rank = 0.4). Creatinine levels in the group of patients whose FBXM was positive (1.4 +/- 0.4 mg/dL; n = 76) were not significantly different from the group with a negative FBXM (1.4 +/- 0.4 mg/dL; n = 42). Even in the presence of class II PRA, a positive FBXM did not impact a patient's creatinine levels or graft outcome. CONCLUSION: Neither short nor long-term graft survival of deceased donor kidneys is influenced by a positive flow B-cell IgG crossmatch, even when caused by HLA class II antibody.     

Sensitization and sensitivity: defining the unsensitized patient

BACKGROUND: Since the landmark studies of Patel and Terasaki in the late 1960s, pretransplant cross-matching has been performed by HLA laboratories on a 24-hr/7-day basis. In fact, regulating agencies such as the American Society for Histocompatibility and Immunogenetics and the United Network for Organ Sharing have mandated prospective crossmatching for selected solid organ transplants. However, two recent publications (Transplantation 1998; 66: 1833; and Transplantation 1998; 66: 1835) have suggested a change to this approach. Specifically, those authors advocate the transplantation of non-sensitized individuals without a final prospective cross-match as a means to reduce cold ischemia time and the incidence of delayed graft function. Such considerations were predicated upon results generated by cytotoxicity-based antibody screening. We and others, however, have reported that a flow cytometric-based assay is a more sensitive method to detect alloantibodies than cytotoxicity. Furthermore, an increasing number of reports document that graft survival is improved among patients whose final flow cytometric crossmatches were negative compared to patients with positive flow cytometric crossmatches. Although we agree that it is reasonable to transplant truly non-sensitized patients without a prospective final crossmatch, our data demonstrate that a large number of patients deemed non-sensitized by cytotoxicity-based antibody assessment are, in fact, sensitized. METHODS: Panel-reactive antibody (PRA) testing was performed with 703 sera from 527 patients. The patient population consisted of individuals awaiting either renal or cardiac transplantation. PRA evaluations were performed using lymphocyte cytotoxicity (antiglobulin-enhanced, complement-dependent cytotoxicity [AHG-CDC]) or assays (enzyme-linked immunosorbent assay [ELISA]; flow cytometry) in which solubilized HLA molecules were affixed to solid phase matrices. RESULTS: PRA activity in 264 sera from 88 patients was evaluated by AHG-CDC, ELISA, and flow cytometry. Results among the three methods were concordant for 83% of these sera. Discordant results occurred with 32 samples and demonstrated a distinct hierarchy in the sensitivity of the three techniques to detect alloantibodies. None of the 32 sera were positive by AHG-CDC, 20/32 were positive by ELISA, and 32/32 were positive by flow cytometry. Subsequent studies revealed that, among 527 patients, 302 (57%) exhibited 0% PRA by AHG-CDC. Of these 302 AHG-CDC-negative patients, 76 (25%) had class I or class II antibodies detectable using a flow cytometric approach. Within the AHG-CDC-negative/flow cytometric-positive patients, PRA values exhibited a wide range (6-99%) for both class I and class II antibodies. The average PRA was 27% and 38% for class I and II, respectively. Retrospective flow cytometric crossmatches performed for 30 recipients of cardiac allografts whose AHG-CDC PRA were 0% revealed that 11/30 crossmatches were positive. CONCLUSIONS: The concept of transplanting non-sensitized patients without a prospective final crossmatch is appealing and, if bona fide, clearly makes sense. However, our data demonstrate that how a patient is deemed non-sensitized is critical. The difference between AHG- and flow cytometric-based PRA testing is significant and can result in transplantation of alloimmunized patients considered to be non-sensitized. Therefore, we recommend that, if a transplant center chooses to forego a prospective final crossmatch, the decision to do so should be based on methods more sensitive than AHG-CDC.     

Relevance of posttransplant flow cytometric T- and B-cell crossmatches in tacrolimus-treated renal transplant patients

BACKGROUND: De novo development of anti-human leukocyte antigen (HLA) antibodies after transplantation is associated with increased rejection and decreased graft survival. In this study, the effect of posttransplant HLA antibodies on clinical outcome was evaluated in patients treated with tacrolimus by means of flow cytometric crossmatches (FCXm). METHODS: T- and B-cell FCXm were performed retrospectively on posttransplant sera of patients who received a graft between 1997 and 1999. Ninety-four kidney-only recipients were tested and all FCXm positive sera were investigated for the presence of HLA class I and II antibodies by Flow panel reactive antibodies. RESULTS: From 94 patients with a negative pretransplant complement-dependent cytotoxicity crossmatch, seven (7%) showed a positive pretransplant FCXm. After transplantation the FCXm became positive in five patients (6%). The predictive value of a positive FCXm after transplantation, and the log-transformed relative change in fluorescence ratio between pretransplant and posttransplant serum, were not significant to rejection within six months, nor to graft survival censored for death. CONCLUSIONS: The presence of HLA antibodies before rejection or graft failure could only be shown in a minority of patients; most antibodies were detected after graft failure, especially after transplantectomy. Monitoring through antibody testing after transplantation on the basis of our results has no added value with tacrolimus-based immunosuppression.