Reports

Comprehensive reports may include (for CD4 / CD8-sorted T-cells) the following data:

Data acquisition and data analysis. Absolute cell count, CD4/CD8 ratio. Sorting: CD4/CD8 absolute count after sorting. After mRNA isolation and cDNA transcription: Quality control of mRNA / cDNA.

TCR variable gene analysis (for individual VA, VB, VG or VD genes as requested).

TCR CDR3 analysis:

Synopsis of each TCR V family and potentially a three dimensional data report.

Quantification of individual TCR families either by flow-cytometry (see Figure 6) and/or measuring individual TCR mRNA transcripts. A quality control report of the laboratory which addresses the sensitivity and specificity of the TCR CDR 3 analysis, including data for inter- and intra-assay variation.

Monoclonal TCRs should be confirmed by DNA sequence analysis. More than 2 monoclonal TCRs in a healthy individual: Repeat the entire panel, including flow-cytometry, if applicable. The presence or absence of each TCR variable family should be noted. The process of TCR CDR3 analysis is compiled in Figure 7.

Figure 7. Compilation of TCR CDR3 fingerprinting: Amplification of each individual TCR V-family (e.g. here TCR VB2), run-off reaction and measuring the CDR3 length. T-cells may be quantified either by measuring the TCR VB mRNA content, or by flow-cytometry. Comparative analysis will aid to visualize differences in the T-cell pool, e.g. as response to therapy or any intervention affecting the cellular immune system.

Figure 7. Compilation of TCR CDR3 fingerprinting: Amplification of each individual TCR V-family (e.g. here TCR VB2), run-off reaction and measuring the CDR3 length. T-cells may be quantified either by measuring the TCR VB mRNA content, or by flow-cytometry. Comparative analysis will aid to visualize differences in the T-cell pool, e.g. as response to therapy or any intervention affecting the cellular immune system.

If monoclonal TCR sequences are reported: report of the variable family, the joining region, D-region (for VB families) and the exact nucleotide and amino acid sequence of the CDR3 region responsible for TCR specificity.

If flow-cytometric analysis has been implemented to gauge the TCR repertoire:

List of each individual TCR VB family.

Differences in regard of quality (molecular composition) and quantity of each TCR family are reported as percent over- or under-representation as compared to

1. a control sample determined by the investigator in individual patients (i.e. prior or after therapy),

2. different anatomic compartments (e.g. TIL versus PBL), or

3. data from a normal healthy control population.

Selected reasons for false positive results (e.g.'false' monoclonality):

Insufficient number of T-cells may indicate monoclonality: higher cell 'input' will reveal the real picture (Figure 3).

False 'negative results:

Insufficient sorting/purity of T-cells, e.g. a monoclonal TCR in the CD8 population (e.g. in the TCR VB1 family) may be 'masked' by a polyclonal TCR population in the contaminating CD4+ T-cell population. Poor mRNA isolation and quality in the target population / tissue of interest. TCR mRNA 'turnover' may be associated with T-cell activation or resting state.

Specimens suitable for evaluation:

• Fresh frozen tissue (not paraffin-embedded tissue, although CDR3 analysis has been performed using paraffin-embedded tissues (48)

• Synovial fluid

Cerebrospinal fluid

• Material from bronchoalveolar fluid

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