Donna P. Lucas

Naturally occurring interference in Luminex® assays for HLA-specific antibodies: Characteristics and resolution

Substances occurring naturally in the sera of patients can interfere with Luminex antibody assays, causing increased background and changes in antibody specificity. We present data on the effectiveness of hypotonic dialysis (HD) or dithiothreitol (DTT) treatment in eliminating this interference. HD significantly increased reaction strength of positive control beads and reduced reaction strength of negative control beads. HD also improved specificity identification, determination of donor-specific antibody (DSA) strength, and crossmatch predictability compared with values in untreated serum. DTT also increased the reaction strength of positive control beads, but in most cases, further increased reactivity of negative control beads. DTT improved crossmatch predictability but to a lesser extent than did HD and may differ with specificities defined in other assays. Because interference is frequently observed in sera from highly sensitized patients, it is important to recognize and eliminate interference in Luminex antibody assays for accurate and meaningful test interpretation.

Characterization of HLA class I specific antibodies by ELISA using solubilized antigen targets: II. Clinical relevance.

Until recently, the nature of humoral sensitization to HLA has been characterized by data from lymphocyte-based assays, predominantly cytotoxicity tests. We have examined the characteristics, determined by enzyme-linked immunosorbent assay (ELISA), of sera from 191 subjects known to have produced HLA-specific antibody. We found that ELISA detected higher frequencies compared with cytotoxicity of many (74.5%), but not all, HLA-specific antibodies; in many cases (42.6%) the frequencies of these antibodies were higher than predicted from population frequencies whereas some antibodies (23.4%) occurred with lower than expected frequencies. Some of the increase in frequencies can be accounted for by crossreactivity, i.e., sensitization to epitopes shared among two or more allelic products. The presence of epitopes shared between a recipients antigen and a mismatched antigen in a donor also tended to narrow the specificity of antibody produced. However the data also indicate differences in immunogenicity among different antigens suggesting that crossreactive group matching would be beneficial in some but not all cases. Finally, we present case studies to illustrate the value of ELISA in predicting humoral rejection episodes and in monitoring the efficacy of rejection therapies.

Characterization of HLA class I specific antibodies by ELISA using solubilized antigen targets: I. evaluation of the GTI QuikID assay and analysis of antibody patterns

The development of solid phase immunoassays using solubilized HLA molecules as targets has provided a means of detecting HLA-specific antibodies that overcomes many of the shortcomings of lymphocyte based assays. We have evaluated a commercially available assay, the GTI QuikID (QID), that uses solubilized class I molecules from 40 subjects selected for their HLA phenotype, to characterize HLA-specific antibodies. We tested 595 sera from 319 subjects and compared the results obtained with QID to those obtained with cytotoxicity (CYT) and with GTI QuikScreen (QS) as well as to historic data. The correlation of QID with CYT (r = 0.54) was comparable to that between QID and QS (r = 0.60). The majority of disparities between QS and QID were apparent false negatives with QID that could be overcome by analyzing QID data at lower cutoff values. In contrast, most of the disparities between QID and CYT were false negatives in CYT due to the relatively low sensitivity of that assay. As expected, the ELISA was more sensitive (97%) than CYT (78%) but had a somewhat lower specificity (87% vs. 92%) due, most likely, to selection of sera that excluded most sera that were known to be nonspecific by CYT. Determination of antibody specificity could be achieved quickly by manual analysis of the QID data because of the way the data are presented by the manufacturers software. Interestingly, the frequencies of different antibodies detected by ELISA differed from those detected by CYT with ELISA identifying more sera containing antibodies to both A and B locus antigens.

Evaluation of HLA antibodies with the PRA-STAT test : an ELISA test using soluble HLA class I molecules

HLA-specific antibody, present before or after transplantation, may adversely effect graft outcome. Antibody testing by cytotoxicity (CYT) is laborious, requires viable lymphocytes, does not differentiate non-HLA cytotoxic antibody, and cannot be used readily on specimens from patients being treated with cytotoxic antibodies. We have evaluated PRA-STAT, an antibody screening kit that uses an ELISA test with soluble HLA class I molecules as targets. We performed 219 tests on a variety of serum specimens, 128 of which were also tested by CYT. There was a highly significant correlation (r = 0.78, P < 0.001) between PRA-STAT (PS) and CYT for the detection of IgG antibodies. Of 66 sera reactive in both assays, 18% had identical specificities defined in both, 27% were more reactive in PS than in CYT, 8% were more reactive in CYT, and 47% had different specificities in the 2 assays, with overlap in slightly more than half the cases. Of 13 sera reactive only in PS, 2 were from non-transfused, nontransplanted males with no evidence of lymphocyte-reactive antibody by antiglobulin tests. PS uses an IgG-specific conjugate, therefore IgM class I-specific antibodies cannot be identified--however, their presence does affect test outcome. This, as well as the panel composition and interlot reproducibility, are areas we believe need to be addressed. The PRA-STAT system is rapid, does not require viable cells or complement, and can be automated in part. Resolution of the problems identified here and availability of an IgM-specific conjugate should make this test system a valuable tool in histocompatibility testing.

HLA antibody detection and characterization by solid phase immunoassays: methods and pitfalls.

Solid phase immunoassays for the detection and characterization of HLA-specific antibodies provide greatly increased sensitivity, specificity, and time and reagent efficiency, compared to the traditionally used cell-based methods. Testing is performed using commercially available test kits. The assays are of two general types: enzyme-linked immunosorbent assays and multianalyte bead. The types vary in both sensitivity and equipment requirements.While these assays afford great improvement over the cell-based assays, they can be confounded by interference from substances within the serum that result in high background reactivity. The high sensitivity of the assays also makes them more susceptible to environmental factors and operator variability. The user must be aware of the capabilities of the various formats, the factors that can affect test results, and lot to lot variability of any single product. Knowledge of the characteristics of each product and thorough and accurate analysis of the results are essential to the utility of these assays.

Elevated pretransplantation soluble CD30 is associated with decreased early allograft function after human lung transplantation.

Early allograft function after lung transplantation is variable. Clinical criteria have limited predictive value for early graft function. Recipient immunologic state before LTx may affect early lung function. We investigated the association between pretransplantation soluble CD30 (sCD30), a marker of Th2-type T-cell activation, and early clinical parameters of allograft function. Between September 2002 and January 2007, a total of 80 transplantations were performed at Johns Hopkins Hospital. Of the patients, 43 had a pretransplantation sCD30 level determined. Pre- and postoperative patient variables were collected, and patients were stratified into two groups: sCD30 <20 (low sCD30) and >20 (high sCD30). High sCD30 (n = 26) and low sCD30 (n = 17) groups were similar in age, gender, and ischemia time. In the high sCD30 group, a higher percentage of patients had pulmonary fibrosis and a lower percentage had emphysema. Oxygenation at 48 hours was significantly worse in the high sCD30 group as compared with the low sCD30 (p = 0.003). Moreover, prolonged intubation and 90-day mortality were greater in the high sCD30 group. This represents the first report of the use of sCD30 as a marker for early allograft function in human lung transplanation. Increased pretransplantation recipient sCD30 appears to be associated with decreased early post-transplantation gas exchange, prolonged intubation, and early mortality.

A Flow Cytometric Crossmatch Test Using Endothelial Precursor Cells Isolated from Peripheral Blood

Flow cytometric crossmatch tests provide a donor-specific, cell based method for the detection of alloreactive antibodies in the sera of transplant patients. Conventional crossmatch tests used in solid organ transplantation utilize lymphocytes as target cells to detect the presence of alloreactive HLA antibodies. Isolation of endothelial precursor cells (EPCs) from peripheral blood now allows testing for antibodies reactive with non-HLA endothelial cell antigens.

Tetramer Staining for the Detection of HLA-Specific B cells

HLA-specific B cells can be identified, quantified, and isolated after staining with HLA tetramers. Quantification of these B cells can in turn identify individuals who are sensitized to HLA antigens and the isolation of these cells facilitates a variety of experimental investigations.

Characterization of an antibody specific for HLA-A36 and not reactive with HLA-A1

Humoral sensitization to HLA often results in antibodies to public determinants shared among two or more antigens. Although monoclonal antibodies to A36 have been produced, there are no reports of polyclonal antibodies that react with A36 but not A1. We report here sera from a heart transplant recipient that reacted with A36 but not A1 in tests with both phenotype and single antigen panels on the Luminex platform. Flow cytometric crossmatch tests yielded positive results with an A36 bearing phenotype but not with a phenotype containing A1. A36 reactivity in solid phase assays was abrogated by absorption with cells bearing A36, but not with A1-positive cells. The frequency of B cells in this patient specific for A1 was comparable to that for individuals not sensitized to A1. These data indicate that reactivity was to an epitope present on A36 but absent from A1.