Serological HLA typing

HLA typing by serology detects polymorphism on HLA molecules expressed on the lymphocyte cell surface. The standard method for serological detection is the micro-lymphocytotoxicity assay (5). This assay involves testing the HLA molecules on lymphocytes against a panel of antisera or monoclonal antibodies, whose specificity for HLA polymorphism has been well characterized. If these antisera or monoclonals bind to the HLA molecules on the cell, they induce lysis in the presence of complement (see Figure 3). This requires the antisera to be of the appropriate class and subclass of immunoglobulin (IgGl, IgG3, IgM) for complement activation.

The serological typing of HLA antigens has developed as a unique method used by specialized laboratories. A consequence of the development of this method has been the requirement for dedicated equipment (adapted fluorescence microscopes, Terasaki typing plates, Hamilton syringes), specific

Micro-lymphocytotoxicity test

Micro-lymphocytotoxicity test

Lymphocytotoxicity Test Hla

Figure3. The components of the micro-lymphotoxicity assay are shown in this figure. Viable peripheral blood mononuclear cells (class I) or separated B cells (class II) are mixed with antisera of known HLA specificity. If antisera is bound to a cell (a), this mediates the activation of complement, which is added to the assay. Complement in the presence of the appropriate immunoglobulin class of antisera will puncture the cell (b). Dye can then enter the dead cell denoting a positive result (c), and this can be visually detected through an appropriate microscope. The absence of dye within the cell denotes a negative result and indicates that the specificity of the antisera is different to that expressed by the cells tested.

Figure3. The components of the micro-lymphotoxicity assay are shown in this figure. Viable peripheral blood mononuclear cells (class I) or separated B cells (class II) are mixed with antisera of known HLA specificity. If antisera is bound to a cell (a), this mediates the activation of complement, which is added to the assay. Complement in the presence of the appropriate immunoglobulin class of antisera will puncture the cell (b). Dye can then enter the dead cell denoting a positive result (c), and this can be visually detected through an appropriate microscope. The absence of dye within the cell denotes a negative result and indicates that the specificity of the antisera is different to that expressed by the cells tested.

reagents (panels of highly characterized antisera, panels of highly characterized cell lines). Additionally, experienced technicians, knowledgeable in the use, characteristics, and specificity of HLA typing reagents are required to perform the assay successfully. For this reason, HLA typing has, for the main part, remained within the domain of the professional histocompatibility testing laboratory.

Serological HLA typing has been the principal method for tissue-typing since the 1960s. It was first developed by Terasaki and McLelland in 1964 (5), and has been modified over the years (39). It remains the method of choice in many laboratories, although its current use is mostly restricted to HLA class I, since serological typing for HLA class II offers poor resolution, and is made more complex through several requirements additional to those for class I typing. These include the isolation of class II-expressing B cells from the peripheral blood mononuclear cell (PBMC) populations, and purging antisera of class I specificities. Class II typing is currently performed by PCR-based methods in most histocompatibility laboratories.

A thorough description of all that serological typing entails would be beyond the scope of this chapter. What will be described here are the rudiments of the method, highlighting certain crucial points that must be addressed to successfully typing by serology.

The assay involves three main steps:

(a) the preparation of component reagents, including the separation and storage of PBMCs;

(b) the incubation of cells with sera and complement; and

(c) the detection of cell lysis and the interpretation of results.

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Responses

  • gabriele
    How is complement detected in hla testing?
    8 years ago
  • riccardo endrizzi
    WHO antigen specificity serological?
    7 years ago
  • bobby
    What is serological HLA typing?
    7 years ago
  • teuvo karjalainen
    How is hla serological typing performed?
    7 years ago
  • elise
    What is the serological tissue typing test used for?
    1 year ago

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