Daniel S. Ramon

The complexity of human leukocyte antigen (HLA)-DQ antibodies and its effect on virtual crossmatching.

Background. We previously reported that in patients possessing human leukocyte antigen (HLA)-DQ-directed antibodies, the target molecule may include the patient’s own DQ&bgr; chain if it is paired with non–self-DQ&agr; chain, thus forming a different DQ target. Herein, we sought to assess the breadth of this phenomenon. Methods. Serum samples from 104 patients awaiting kidney transplantation, known to have DQ antibodies, were studied. Antibody identification was performed using luminex-based HLA class II single-antigen bead assays from two vendors; DQA1/DQB1 typing was performed using luminex polymerase chain reaction – sequence specific oligo prob hybridization (PCR-SSO) technology. Results. A total of 71% of the 104 serum samples studied contained antibodies reactive against test beads coated with the patient’s own DQ&agr;- or &bgr;-chain components. Of those, 35 patients (34%) exhibited antibodies to their own DQ&bgr; chain when in combination with non–self-DQ&agr; chains; and 64 patients (62%) had antibodies to their own DQ&agr; chain when in combination with non–self-DQ&bgr; chains. This is a striking observation. Conclusions. To the best of our knowledge, this is the first systematic, high-resolution evaluation of DQ antibody repertoire. With the expansion of virtual crossmatching, particularly in the context of a national registry, the need for more detailed DQ antibody or antigen evaluation is critical to improve operational efficiency and patient outcomes.

Perception versus reality ?: Virtual crossmatch - How to overcome some of the technical and logistic limitations

The goal of this work was to evaluate concordance between (a) actual flow cytometric crossmatch (FCXM) that is performed by the OPO laboratory servicing our transplant center and (b) virtual XM (vXM) prediction based on antibody identification by solid‐phase methods performed in our laboratory.

Pyrosequencing™ : A one-step method for high resolution HLA typing

While the use of high-resolution molecular typing in routine matching of h uman l eukocyte a ntigens (HLA) is expected to improve unrelated donor selection and transplant outcome, the genetic complexity of HLA still makes the current methodology limited and laborious. Pyrosequencing™ is a gel-free, sequencing-by-synthesis method. In a Pyrosequencing reaction, nucleotide incorporation proceeds sequentially along each DNA template at a given n ucleotide d ispensation o rder (NDO) that is programmed into a pyrosequencer. Here we describe the design of a NDO that generates a pyrogram unique for any given allele or combination of alleles. We present examples of unique pyrograms generated from each of two heterozygous HLA templates, which would otherwise remain cis/trans ambiguous using standard s equencing b ased t yping (SBT) method. In addition, we display representative data that demonstrate long read and linear signal generation. These features are prerequisite of high-resolution typing and automated data analysis. In conclusion Pyrosequencing is a one-step method for high resolution DNA typing.

Antibodies against HLA-DQ α-chain and their role in organ transplantation

The availability of solid phase based testing for the detection of antibodies to HLA antigens has significantly increased the sensitivity and specificity of antibody identification. However, it is important to understand the nature and complexity of the HLA system and of the reagents used for its identification in order to accurately interpret solid phase based testing results. Here we present two case reports; in the main case presented, the findings may have led to denying a patient from receiving an immunologically compatible organ, if careful interpretation of antibody testing results had not been performed. Interestingly, those were antibodies directed at the alpha-chain (or combination of alpha- and beta-chain) of a DQ antigen.