Lower Median Fluorescence Intensity Research Articles

Specificity, strength, and evolution of pretransplant donor-specific HLA antibodies determine outcome after kidney transplantation.

In this cohort study (N=924), we investigated the evolution and clinical significance of pretransplant donor-specific HLA antibodies (preDSA), detected in the single-antigen beads assay but complement-dependent cytotoxicity crossmatch-negative. Donor specificity of the preDSA (N=107) was determined by high-resolution genotyping of donor-recipient pairs. We found that in 52% of the patients with preDSA, preDSA spontaneously resolved within the first 3months posttransplant. PreDSA that persisted posttransplant had higher pretransplant median fluorescence intensity values and more specificity against DQ. Patients with both resolved and persistent DSA had a high incidence of histological picture of antibody-mediated rejection (ABMRh ; 54% and 59% respectively). Patients with preDSA that persisted posttransplant had worse 10-year graft survival compared to resolved DSA and preDSA-negative patients. Compared to cases without preDSA, Cox modeling revealed an increased risk of graft failure only in the patients with persistent DSA, in the presence (hazard ratio [HR]=8.3) but also in the absence (HR=4.3) of ABMRh . In contrast, no increased risk of graft failure was seen in patients with resolved DSA. We conclude that persistence of preDSA posttransplant has a negative impact on graft survival, beyond ABMRh . Even in the absence of antibody-targeting therapy, low median fluorescence intensity DSA and non-DQ preDSA often disappear early posttransplantation and are not deleterious for graft outcome.

Assessing the correlation between C3d-binding de novo donor specific HLA antibodies and rejection in kidney transplant recipients

Aim Solid phase assays have the ability to detect even low levels of HLA antibodies, but potentially lack the ability to distinguish between clinically relevant or irrelevant DSA. Here we test the clinical significance of a novel C3d binding assay. Methods Serum samples from 85 kidney transplant recipients were retrospectively analyzed using the Immucor© Single Antigen immunoassays, to detect either IgG or C3d binding HLA antibodies. Inclusion criteria included patients that developed de novo DSA by Luminex One Lambda (OL) single antigen assay and had a biopsy obtained at the same time point as the serum sample. Median fluorescence intensity (MFI) value of 1000 was arbitrarily used to assign positive responses. Results Of the 85 patients: 21 had AMR, 12 cell-mediated rejection (CMR), 11 mixed rejection (AMR + CMR), 4 with pathologic finding other than rejection, such as BK virus, and 41 had normal biopsies. C3d DSA were detected in 16/85 patients with the majority having AMR in 11/16 patients (69%) compared to 1/16 patients (6%) with CMR, 2/16 patients (12.5%) with a mixed rejection, and 2/16 patients (12.5%) with negative biopsies. These 2 negative biopsy patients had strong MFI values for Immucor IgG DSA and strong titers on the OL platform; moreover, both of these patients went on to have AMR later on. Immucor IgG assay detected 29/85 patients with DSA, in which, the majority of C3d positive DSA (16/16) being Immucor IgG class II DSA-positive. Immucor IgG positive DSA with negative C3d results had lower MFI (Fig. 1). All the C3d positive assays corresponded to moderate to high titer OL results (titers ⩾ 1 : 64 ), although three samples had class II specificities with titers ⩾ 1 : 128 were not C3d positive. Conclusion C3d-binding antibodies seem to be associated with stronger antibodies with at least moderate level titer and rejection. Further studies are required to substantiate our results. Download : Download full-size image

P32 Prospective study on human papillomavirus significance in oral dysplasias in association with clinical parameters and P16INK4a expression

Human papillomavirus (HPV) DNA and RNA is found in a subset of oral cavity squamous cell carcinomas (OSCC), indicating a transforming role of the virus in a proportion of those cancers. HPV DNA has also been detected in some oral premalignant lesions (OPL). However, the significance of HPV DNA detection in those lesions is currently unclear. In particular, the association with clinical parameters, p16INK4a and progression risk has not been analyzed in a prospective cohort yet. Formalin-fixed, paraffin-embedded biopsies of mild, moderate and severe OPL deriving from the “Oral Cancer Prediction Longitudinal study” (OCPL), British Columbia, Canada, were analyzed for HPV DNA, genotype and semi-quantitative viral load applying Luminex technology. The results were correlated to various clinical parameters including time to progression and p16INK4a immunohistochemical expression as a potential surrogate marker of HPV-induced transformation in those lesions. Thirteen out of 149 (8.7%) samples were HPV DNA-positive (high risk (HR)-HPV16: 7/13, HR-HPV18: 6/13) with a low mean “median fluorescence intensity (MFI)” as a semi-quantitative measure of viral load (204.6 and 186.8 MFI, respectively). HPV DNA-positivity was associated with higher lesion progression risk (p = 0.038). Half of the 149 samples demonstrated focal p16INK4a expression (52%), 13% showed a diffuse pattern and 35% were p16INK4a-negative. Staining patterns did not differ by age, gender, ethnicity or smoking habit (ever vs never smoker); however, current smokers more often showed p16INK4a overexpression (37% focal, 42% diffuse, 17% negative; p = 0.02). Progression occurred in 7 lesions: 0/78 focal, 6/52 negative and 1/19 diffuse p16INK4a expression (p = 0.01). No significant association between HPV DNA-positivity and p16INK4a expression was observed. Our results demonstrate a higher progression risk in HPV DNA-positive OPL patients. HPV DNA-positivity in those lesions might represent a non-transforming bystander infection, particularly considering the comparatively low MFIs and the lacking association with p16INK4a expression. Assessment of HR-HPV oncogene transcription in the HPV DNA-positive OPL will be performed to differentiate truly HPV-transformed lesions from potential bystander infections. OPL with focal p16INK4a expression appear to have a low likelihood of progression independent of HPV DNA status.

Epitope-specificities of HLA antibodies: The effect of epitope structure on Luminex technique-dependent antibody reactivity

The search of HLA antibodies is currently more accessible by solid-phase techniques (Luminex) in the immunized patients leading to an expansion of the antibody patterns. The aim of this study was to investigate low median fluorescence intensity value in unexpected reactivity patterns. Here, we performed HLAMatchmaker analyses to evaluate the potential functional epitopes that can elicit HLA-specific alloantibody responses in a pregnancy-sensitized woman with an epitope defined by the 82LR. Surprisingly, in according to the registry of HLA epitopes, we found that 82LR epitope covered all allelic specificities of our unexpected antibody patterns, shared between Bw4-positive HLA-B antigen and HLA-A23, -A24, -A25 and -A32. This finding is consistent with the verification of HLA ABC epitope recorded in the website-based HLA Epitope Registry and addresses the importance of determining HLA antibody epitope-specificities on Luminex technique-dependent antibody reactivity.

Low median fluorescence intensity could be a nonsafety concept of immunologic risk evaluation in patients with shared molecular eplets in kidney transplantation

Human leukocyte antigen (HLA) antibodies are usually "epitope" and not "antigen" specific. This work presents an interesting case concerning Luminex median fluorescence intensity (MFI) levels in antibodies considered low risk (<1,000), but producing humoral rejection. These low-titer antibodies could play an important role in transplantation. A 42-year-old woman was retransplanted with a deceased donor with negative complement-dependent cytotoxicity cross-matching. Our patient was pretransplant (PrT) sensitized to HLA antigens (single antigens (SA) = 31%) for 1 previous transplant. Thus, the formerly detected sensitized antigens were A32, A30, A31, cross-reacting group 5C, and DQ3 with a MFI(max) ≈ 4,127. In the posttransplantation period (PTP), the patient exhibited important instability in renal function and we detected an increased SA percentage (61%) with MFI(max) = 15,029 (A*32) with other antigens (detected with a low PrT MFI [<1,000]) as anti-A*03 (MFI(max) = 13,301) and anti-A*11 (MFI(max) = 13,714) specificities. Anti-A*03 was a donor-specific antibody (DSA). Renal biopsy was compatible with humoral rejection. The patient was pulsed with methylprednisolone, plasmapheresis, and intravenous immunoglobulin without improvement. Thus, we added anti-CD20 and the initial clinical response was highly favorable. Biopsies resulted in suggestive rejection reversion. MFI A*03 DSA decreased to 6,908 and later to MFI(max) = 5,505. After a 6-month PTP, the patient is well with MFI(max) = 3,124. It was possible to define exactly the potential immunizing epitope eplets whose recognition determined the specific antibody production. A*32:01, A*30:01, A*31:01 (detected PrT), A*11:01, and A*03:01 (detected PTP) alleles have several shared eplets (62QE, 70AQS, and 76VGT), with 62QE being the only eplet present on all alleles. In conclusion, low MFI levels in antibodies considered low risk could be important in posttransplant humoral rejection, although the patient's renal function can be restored. Thus, specific shared eplets should always be investigated with respect to previous transplant mismatches.

Comparative density of the human T-cell antigen T65 on normal peripheral blood T cells and chronic lymphocytic leukemia cells

A 65,000 dalton T-cell specific antigen previously demonstrated to be present on the surface of normal and malignant T cells, but not normal B cells, has been detected on the surface of leukemic cells from patients wih nonsecretory, surface immunoglobulin-positive chronic lymphocytic leukemia (CLL). By means of immunofluorescence and flow cytometry, the relative surface density of the T65 antigen on CLL cells was compared to that on normal peripheral blood T cells and human thymocytes, as well as cell lines of T-cell lineage. In all cases, the CLL cells had a more homogeneous and a lower median fluorescence intensity than that of normal circulating T cells. Thymocytes were composed of three populations, two with low surface density of T65 resembling the CLL cells and the other with higher density similar to normal T cells. The staining of cell lines varied from bright, heterogeneous staining (8402) to uniform, low-density staining (Molt- 4). The implications of these findings with regard to lymphocyte differentiation are discussed.

Comparative Density of the Human T-Cell Antigen T65 on Normal Peripheral Blood T Cells and Chronic Lymphocytic Leukemia Cells

AbstractA 65,000 dalton T-cell specific antigen previously demonstrated to be present on the surface of normal and malignant T cells, but not normal B cells, has been detected on the surface of leukemic cells from patients with nonsecretory, surface immunoglobulin-positive chronic lymphocytic leukemia (CLL). By means of immunofluorescence and flow cytometry, the relative surface density of the T65 antigen on CLL cells was compared to that on normal peripheral blood T cells and human thymocytes, as well as cell lines of T-cell lineage. In all cases, the CLL cells had a more homogeneous and a lower median fluorescence intensity than that of normal circulating T cells. Thymocytes were composed of three populations, two with low surface density of T65 resembling the CLL cells and the other with higher density similar to normal T cells. The staining of cell lines varied from bright, heterogeneous staining (8402) to uniform, low-density staining (Molt-4). The implications of these findings with regard to lymphocyte differentiation are discussed.