Long-term Outcome of Allogeneic Hematopoietic Stem Cell Transplantation From Unrelated Donor Using Tacrolimus/Sirolimus-based GvHD Prophylaxis: Impact of HLA Mismatch.

BACKGROUND: While tacrolimus and sirolimus (T/S)-based graft-versus-host disease (GvHD) prophylaxis has been effective in preventing acute GvHD post hematopoietic cell transplantation (HCT), its efficacy and long-term outcome in matched (MUD) and mismatched unrelated donor (mMUD) setting is not well defined. METHODS: Herein, we evaluated a consecutive case-series of 482 patients who underwent unrelated donor HCT (2005-2013) with T/S-based GvHD prophylaxis. RESULTS: With a median follow-up of 6.2 years (range = 2.4-11.3), the 5-year overall survival (OS) and relapse/progression-free survival were 47.5% (95% confidence interval [CI]: 43.0-52.0) and 43.6% (95% CI: 39.1-48.1), respectively; and the 5-year cumulative incidence of nonrelapse mortality (NRM) and relapse were 24.9%, and 31.5%, respectively. In this cohort, mMUD was associated with worse OS (39.0% versus 50.7% at 5 y; P = 0.034), primarily due to greater risk of NRM (33.5% versus 21.7%; P = 0.038). While rates of relapse, acute (II-IV or III-IV) or chronic GvHD (limited or extensive) were not different, death caused by chronic GvHD (20.8% versus 12.8%; P = 0.022) and infection (33.0% versus 18.1%; P < 0.01) were significantly greater in mMUD. In multivariable analysis, high-risk disease (hazard ratio [HR] = 2.21, 95% CI: 1.16-4.23; P < 0.01) and mMUD (HR = 1.55, 95% CI: 1.15-2.08; P = 0.004) were independent predictive factors for OS. CONCLUSIONS: T/S-based GvHD prophylaxis is an effective and acceptable GvHD prophylactic regimen. However, survival after mMUD remained poor, possibly related to the severity of chronic GvHD.

EDTA Treatment for Overcoming the Prozone Effect and for Predicting C1q Binding in HLA Antibody Testing.

The Luminex-based single antigen bead (SAB) assay is widely used to detect HLA antibody in transplant recipients. However, one limitation of the SAB assay is the prozone effect, which occurs mostly as a result of complement interference. We investigated the efficacy of EDTA treatment for overcoming the prozone effect and predicting C1q binding of HLA antibody. We subjected 27 non-treated (naïve) and EDTA-treated serum samples from highly sensitized patients to IgG-SAB assays, and we confirmed the prozone effect in 53% and 31% of class I and class II antibody tests, respectively, after EDTA treatment. When we conducted additional assays after dithiothreitol treatment and serum dilution, EDTA was the most efficacious in eliminating the prozone effect. Reducing the prozone effect by EDTA treatment strengthened the correlation between IgG mean fluorescence intensity (MFI) and C1q MFI values (ρ=0.825) as compared with the naïve sera (ρ=0.068). Although C1q positivity was dependent on the concentration of HLA antibody in EDTA-treated sera, the correlations varied individually. Overall, our results confirmed the efficacy of EDTA treatment for overcoming the prozone effect. EDTA treatment showed a positive effect on the correlation between IgG MFI and C1q MFI values.

Comprehensive assessment for serum treatment for single antigen test for detection of HLA antibodies.

The single antigen test is widely used in the field of transplantation to determine the specificity of HLA antibodies. It will be beneficial to standardize the procedure of the single antigen test among HLA laboratories. It is not uncommon that single antigen testing on native sera fails to detect antibodies with very high concentrations. It has been shown that cleavage products of activated complement components may mask strongly binding antibodies in single antigen testing. To overcome inhibition by the activated complement products, sera are pretreated with ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), or heat inactivation before single antigen testing. However, no studies have been published to systemically compare the impact of these treatments on single antigen testing. The aim of this study is to understand the different effects these treatments may have on single antigen test results. We found that mean fluorescence intensity (MFI) obtained from sera treated with EDTA and heat inactivation were nearly identical, while DTT treatment was less potent to remove the inhibition. In addition, sera dilution did not further increase MFI of antibodies after EDTA treatment. Our results provide guidance to choose a pretreatment reagent for single antigen testing, and to compare studies obtained from laboratories using different treatments.

EDTA is superior to DTT treatment for overcoming the prozone effect in HLA antibody testing.

A limitation of solid-phase human leukocyte antigen (HLA) antibody assays is the falsely low/negative result of samples with high-titer antibodies, a phenomenon known as the prozone effect. Here we compared the efficacy of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) treatment of serum samples in overcoming the prozone effect. A total of 21 serum samples were treated with either EDTA or DTT before HLA single antigen bead assay. The efficacy of prozone effect reversal, compared with untreated samples, was examined on fourfold, serially diluted samples, from neat to 1:256, using PBS as diluent. EDTA reversed the prozone effect in all tested samples, with an efficiency of greater than 84%, estimated by the ratio of undiluted sample mean fluorescence intensity (MFI) to peak MFI, for any given dilution. In contrast, the efficiency of DTT treatment was as low as 47%. These results show superior prozone effect reversal with EDTA treatment, compared with DTT.

EDTA Treatment of Serum Unmasks Complement-Mediated Prozone Inhibition in Human Leukocyte Antigen Antibody Testing.

OBJECTIVES: Luminex-based single-antigen bead human leukocyte antigen (HLA) antibody testing is widely used to define HLA antibodies for transplant compatibility. False-negative results can occur with complement-mediated prozone inhibition. This study assessed the effect of EDTA on the assay background reactivity and fluctuations in antibody mean fluorescent intensity. METHODS: Serum specimens were retrospectively tested using Luminex-based single-antigen beads with and without EDTA. Treated and untreated serum samples were compared by two measures: changes in background reactivity and changes in HLA antibody strength after EDTA treatment. RESULTS: Ten pretransplant and 48 posttransplant specimens were identified: lung (22), heart (10), kidney (21), heart/lung (two), pancreas (one), small bowel (one), and liver (one). After EDTA treatment, weak antibodies (below 2,000 mean florescent intensity) demonstrated the largest fluctuations. Newly identified HLA antibodies were seen in 16% (8/49) of class I and 26% (15/57) of class II beads. EDTA treatment did not result in false-negative reactions compared with untreated serum. CONCLUSIONS: EDTA serum pretreatment mitigated complement-mediated prozone inhibition and improved accurate HLA antibody detection. The background reactivity and the false-negative rate of the assay appear unchanged.

[Pitfalls in interpreting anti-HLA antibodies by Luminex technology]. / Pièges de l'interprétation des anticorps anti-HLA par technologie Luminex.

The Luminex technology has become an important tool for HLA antibody screening and identification. This is the most sensitive technology to detect HLA antibodies for transplant patients and patients on awaiting list, and it has ushered a new strategy to determine HLA compatibility between donor and recipient. Moreover, the clinical relevance of all detected anti-HLA antibodies is not well understood, because this technique was shown to be prone to many artefacts or interferences, leading to a complicated interpretation for biologists and clinicians. Our objective in this article is to provide a careful consideration about this solid phase assay, and to focus attention on raised questions about technical performance and interpretation of the results. We should keep in mind that our results could change the clinical management of sensitized patients, their aptitude to receive a graft, and their follow-up.

HLA typing with sequence-specific oligonucleotide primed PCR (PCR-SSO) and use of the Luminex™ technology.

The hybridization products obtained by PCR using sequence-specific oligonucleotides can be traced either by colorimetric (streptavidin-biotin)-, X-ray (digoxigenin-CSPD)-, or fluorescence (FITC, PE)-based detection systems. To achieve a faster, reliable, automated typing technique microbead and fluorescence detection technology have been combined and introduced to this field (XMAP™ technology). For each locus, a series of microspheres, which are recognizable by their specific color originating from two internal fluorescent dyes, are used. Each microsphere is coupled with a single probe that is capable of hybridizing with the biotin-labeled complementary amplicon. Once hybridization occurs, it can be quantified by measuring the fluorescence signal originating from fluorescently (streptavidin-PE) labeled amplicons captured by the beads. Currently, there are two commercially available systems that differ in the scale of probes and the methods used for amplification and denaturation. One of these is described in detail in this chapter.

HLA typing with sequence-specific oligonucleotide primed PCR (PCR-SSO)and use of the Luminex technology.

The hybridization products obtained by PCR using sequence-specific oligonucleotides (PCR-SSO) can be traced either by colorimetric- (streptavidin- biotin), X-ray-(digoxigenin-CSPD), or fluorescence- (FITC, PE) based detection systems. To achieve a faster, reliable, automated typing, microbead and fluorescence detection technology have been combined and introduced to this field (XMAP technology). For each locus, a maximum of 100 microspheres, which are recognizable by their specific color originating from two internal fluorescent dyes, are used. Each microsphere is coupled with a single probe that is capable of hybridizing with the biotin labeled complementary amplicon. Once hybridization occurs, it can be quantified via the fluorescence signal originating from fluorescently (Streptavidin-PE) labeled amplicons captured by the beads. Currently, there are two commercially available systems that differ in the scale of probes and the method of amplification or denaturation. One of these will be described in detail in this chapter.