Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5943_Библиотеки_им_академика_М_И_Перельмана

individual patients showed symptoms of complement activation and acute kidney injury[18, 19].

trials to evaluate the immune response to the therapy. Additionally, there are differ­ent ELISPOT variations. In the related FluoroSpot, the detection antibody is conju­gated with a fluorescent dye[26–29]. The advantage of FluoroSpot over ELISPOT is that it allows for multiplexing, meaning multiple cytokines of one stimulated T cell can be detected simultaneously.

Intracellular cytokine staining (ICS) is a flow cytometry‐based assay that meas­ures the production of cytokines by T cells in response to stimulation with specific antigens[30–33]. The assay involves the stimulation of T cells with the antigen of interest and the addition of Brefeldin A, which inhibits the secretion of various cytokines on a single‐cell basis. The cells are then stained with fluorescently labeled antibodies to identify the specific T‐cell subsets and the cytokines pro­duced. This assay is sensitive and allows to measure polyfunctional T cells, making it an ideal method for assessing the T‐cell response to gene therapies. However, the ICS assay is considered less sensitive than the ELISPOT assay[30–32, 34].

Tetramer staining is a flow cytometry‐based assay that uses fluorescently labeled major histocompatibility complex (MHC) class I tetramers loaded with specific antigenic peptides to detect antigen‐specific T cells[35]. The tetramers bind to the T‐cell receptors (TCRs) of antigen‐specific T cells and can be detected using flow cytometry. This method is specific and considered to have similar sensitivity as the ICS assay and can be used to detect low‐frequency T cells that are able to bind the peptide of interest[36].

the secretion of multiple cytokines from T cells, providing a more comprehen­sive analysis of the T‐cell response to gene therapies. However, it must be con­sidered that cytokines can also be produced by other potentially co‐cultured cells. This has to be taken into account additionally, in context of the frequency of antidrug‐specific T‐cell responses, to avoid data misinterpretation.

immune monitoring in multicenter clinical trials and obtain reproducible results. It has been demonstrated that the lack of reproducibility of methods such as ICS, ELISPOT, and tetramer detection originates from a lack of assay standardization rather than from inherent difficulties in working with live cells and complex assay systems. However, sample and material quality contribute significantly to the quality of results.

In general, other methods can be chosen to assess anti‐AAV therapy T‐cell responses, depending on the scope of the study and the frequency of specific T‐cell responses. In fact, when standardized assays are used in the same samples, ELISPOT results have been shown to correlate with the results of other methods, such as cytokine flow cytometry[46].

validation should be planned for each antigen and cytokine readout to be evalu­ated. Validation approaches for cytokine and recall antigen‐specific ELISPOT assays have been discussed and published in the scientific community[34] and recommendations provided by the Global CRO Council for Bioanalysis (GCC)[48].

Reproducibility (laboratory‐to‐laboratory) is usually excluded from the valida­tion plan, as no other laboratory is involved. However, laboratory‐to‐laboratory precision data have been evaluated and published[44]. The data clearly showed that reproducible and precise data were obtained by ELISPOT‐inexperienced investigators when following standardized procedures and using standardized samples and materials. All laboratories that participated could detect the recall responses within three standard deviations (SDs) of the used reference values.

the antibodies used to capture and detect IFN‐gamma. The cell population tested in ELISPOT assays is specific toward a certain antigen. The assay determines whether the sample includes cells with specificity to the test antigen. The secre­tion of the cytokine of interest is only induced and captured on the ELISPOT plate if the cells are specific to the tested antigen. Cells not activated by the test antigen will not secrete the tested cytokine. The ability of the ELISPOT assay to determine the antigen‐specific and mitogen‐specific lymphocyte responses depends on the antigens employed. The ability of the assay to identify IFN‐gamma from antigen‐ stimulated cells versus spontaneously secreted cytokine is controlled by including cells exposed to medium only for each sample, referred to as medium control.

number of spots that can be counted in that specific assay system, not reaching ‘“too numerous to count” (TNTC) by the used ELISPOT image analyzer.

Exceptions from the bioanalytical method validation guidance are commonly made for ELISPOT validation for accuracy, linearity, and reproducibility. Accuracy is defined as the closeness of agreement between the value determined by the assay and the value that is accepted as a true value or an accepted reference value. As no true reference values are available for this assay, accuracy can be deter­mined by performing measurements employing two different test methods. The same test samples may be used for the IFN‐gamma ELISPOT assay and an orthog­onal method, IFN‐gamma ICS. Both IFN‐gamma measurements can provide the percentage of responding cell population to the same antigen. This percentage or frequency measurement allows comparison of values generated by the two systems. The test sample used for these experiments, however, must have high frequencies as measured in ELISPOT (e.g. more than 400 spots to a given antigen in 400,000 cells per well) to qualify for testing by ICS and for accuracy validation. Accuracy can be determined, but it is not useful for ELISPOT assays in a clinical setting as not enough cells are available from the clinical samples to evaluate accuracy by the proposed methods in a routine manner, and the response fre­quency to the individual antigen is not known. Therefore, accuracy is usually not evaluated. Moreover, determination of accuracy is not necessary because ELISPOT is not a quantitative assay. It does not quantify the amount of IFN‐gamma pro­duced but reports the results as number of positive or no spots counted that pro­duced IFN‐gamma if stimulated by the recall antigen. It is also expected that different tested individuals react differently to the same antigen. However, it has been demonstrated that the ELISPOT assay can be performed accurately, using the above described assay systems[30, 31, 56–58].

Linearity is the ability of the assay to return values that are directly proportional to the concentration of analyte in the sample. Mathematical data transformations to promote linearity may be allowed if there is scientific evidence that the

transformation is appropriate for the method. In general, linearity of the ELISPOT method has been explored in multiple scientific publications, providing evidence for the single‐cell resolution of the ELISPOT assay[30, 31, 56–58]. Linearity can be evaluated, however, is not suited for the clinical sample testing as it is cumber­some, expensive, and requires a lot of cell material and antigens, which is unprac­tical or even impossible to obtain. Moreover, determination of linearity is not required, considering that negative and positive controls are used in the ELISPOT assay run for each clinical sample tested. The medium control commonly employs culture medium with the PBMC samples without antigen stimulation, and the positive control employs a mitogen such as phytohemagglutinin for each sample tested. These control samples are tested in parallel and as a reference for compari­son to the antigen recall (cell and antigen) sample.

Inaddition to T cells, such observations have also been reported for antigen‐ specific B‐cell readouts from cryopreserved PBMCs[64]. In addition to the logistics, sepa­ration, and freezing processes, optimal thawing procedures of the cryopreserved PBMC should be used, making the handling of PBMCs complex and requiring trained personnel to do so, in order not to jeopardize the results of the ELISPOT assay and with it the clinical trial it is supporting[65].

Figure 11.3 demonstrates that whole blood, handled appropriately (using serum‐free solutions) and shipped at room temperature before processing to cryo­preserved PBMCs, can maintain functionality compared with freshly processed blood. There was no statistical difference in recall antigen‐specific IFN‐gamma ELISPOT assays of cryopreserved PBMCs from whole blood kept at room tem­perature when PBMCs were separated immediately or at 6, 12, and 24 hours, con­firming previously published results[55].