Successful outcome of acute graft-versus-host disease in a liver allograft recipient by withdrawal of immunosuppression.

BACKGROUND: Graft-versus-host disease (GVHD) after liver transplantation is uncommon, and the outcome is almost always fatal. Since 1987, about 30 cases have been described, and patient survival is mostly exceptional. METHODS: A 29-year-old man underwent retransplantation due to chronic cholestatic syndrome, 5 years after his first liver transplantation. Indication for the first liver transplantation was acute liver failure caused by exsiccosis. After the second transplantation, the patient had an initially uneventful course, developing thrombocytopenia at day 21 followed by skin rash and septic complications. Diagnosis of acute GVHD was made by using serological techniques for HLA-A and HLA-DRB and subsequently by fluorogenic sequence-specific primed polymerase chain reaction. In addition, donor lymphocytes were marked by immunohistochemical methods via biopsies of the skin. Immunosuppressive therapy was withdrawn to allow the patient's own immune system to eliminate donor cells. RESULTS: By withdrawing the immunosuppressive therapy, clinical and morphological signs of GVHD vanished. The patient is doing well without recurrence 13 months after transplantation. CONCLUSION: Withdrawal of immunosuppressive therapy is a promising approach in the treatment of acute GVHD to allow the patient's immune system to reconstitute itself, reject offending lymphocytes, and avoid lethal septic complications.

HLA-DRB fluorotyping by dark quenching and automated analysis.

In fluorescence-based sequence-specific primed polymerase chain reaction (PCR), referred to as fluorotyping for HLA typing, a major problem is the overlap of the emission spectra of the single dyes. In order to increase the robustness of the previously described HLA-DRB1,3,4,5 low-resolution fluorotyping method, we have constructed two probes quenched by the non-fluorescent acceptor dye DABCYL. The HLA-DRB-specific probe was labeled with FAM, and the internal control probe with TAMRA, respectively, as reporter fluorescent dyes. TAMRA was replaced by DABCYL as a quencher, which led to increased robustness and better discrimination between negative and positive amplification results. ROX was used as a reference to normalize the fluorescence of the reporter dyes. Moreover, as FAM and TAMRA differ strongly by their emission maxima, HLA-DRB-specific and internal control amplification could be clearly distinguished. To further automate data analysis, the software of the TaqMan system 7700 was supplemented by an EXCEL-based calculation table, which directly took over the data. Using modified fluorotyping chemistry and automated data analysis, a total of 201 DNA samples was typed correctly. In summary, HLA-DRB fluorotyping by dark quenching and automated analysis proved to be a robust and reliable tool for research and routine purposes.

Fluorotyping of HLA-A by sequence-specific priming and fluorogenic probing.

The aim of our study was to develop a fluorotyping strategy for the HLA-A locus. In contrast to conventional sequence-specific primed PCR (PCR-SSP), which is based on an agarose gel electrophoresis, fluorotyping eliminates the drawback of low sample throughput, low potential for automation and problems related to contamination. Additionally, fluorotyping is capable of delivering quantitative results depending on the system set-up. The fluorescence-based PCR-SSP assay relies on target-specific and individually labeled fluorogenic probes allowing a simultaneous and differential detection of the specific HLA and the internal control product. The probe used to detect the HLA-A specific amplicons was labeled at its 5' end with 6-carboxyfluorescein (FAM) as the reporter and at its 3' end with 6-carboxy-tetramethylrhodamine (TAMRA) as the quencher. The probe hybridized within the 2nd intron to a conserved region which was found to be identical in all HLA-A alleles and was covered by all primer mixes. During successful PCR the cleavage of the FAM-labeled probe through the 5'-3' exonuclease activity of the Taq DNA-polymerase led to an interruption of the TAMRA-mediated quenching effect and generated a significant increase of the FAM fluorescence. The specific HLA-A typing information was based on the FAM fluorescence released by 24 individual PCR primer mixes. The internal control amplicon was detected with a tetrachloro-6-carboxyfluorescein-TAMRA-labeled fluorogenic probe. Since the HLA-A amplicons had to include the 2nd intron as the target for the fluorogenic probe, the sequence motifs which could be used as priming sites were limited. Therefore, some primer mixes covered more than one specificity resulting in ambiguous amplification patterns in 31 of 231 possible allele or group combinations of HLA-A1-A80. These ambiguities, which all involved the inability to discriminate a particular heterozygous genotype from a homozygous genotype, may be resolved by the quantitative data revealed by fluorotyping. This feature is also helpful to detect new alleles which are not amplified by the current primer mixes.