Frequently Asked Questions:

The process of multiplex detection

1. How many biomarkers can be analyzed in parallel on a tissue section?

One can analyze up to 5-7 biomarkers on a standard 5 micron thick tissue section.  Extra-thick section cuts will support more biomarkers with the use of about 1 micron more thickness per biomarker added.

2. How many fluorescent tags are needed for a multiplex analysis?

Since each biomarker is analyzed on a separate membrane, only one fluorophore is required for all biomarker analyses. To enable cross-normalization between layers, total protein is also measured on each layer in a second fluorescent color, and the intensity ratio is then calculated. This is similar in principle to certain DNA microarray methods. 

3. How does L-IHC compare with multiplex immunofluorescence techniques in which multiple antibodies are applied to the same tissue section?

Standard immunofluorescence is limited to the use of a few fluorophore tags in combination with standard instrumentation. Quantum dot tags offer more colored tags, but their application to tissue sections is hindered by a problem of low tissue penetration due to their large size. In addition,

• Antibodies that target different epitopes in the same target protein (e.g. phospho epitopes) may interfere with one another when applied as a mixture to the specimen. Such steric hindrance cannot occur in L-IHC in which each antibody is applied to a separate specimen surface.
• L-IHC uses only a single flurophore for multiplexed analysis, greatly enhancing ratio-metric evaluations (e.g. phospho vs total),
• L-IHC avoids the problem of tissue autofluorescence
• L-IHC often allows use of the same antibody that works in western blot.

 4. Is a biochemical signal amplification step used to increase the sensitivity?

 Yes. A secondary antibody is applied to the primary antibody on the membrane, followed by a tertiary fluorescent detection reagent.  For further information about the sensitivity and dynamic range of L-IHC, please see the White paper on this website or contact us at MultiplexIHC@2020gene.com.

 5. Does the process of protein transfer through the membrane layers incur any loss of resolution?

 The histomorphology can be clearly resolved, such as cell layers and glandular acini. Single cells are presently at the limit of resolution.   

 6. What is the method used to correlate the biomarker signals on the separated membranes to the histomorphology in original tissue section?

 After transfer, staining and analysis of proteins on the membrane layers, the same tissue section from which the proteins originated can be hematoxylin and eosin stained. The tissue profile signals from the separated biomarker membranes can then be digitally imaged as superimposed onto the H&E section image to correlate signals to regions of interest such as tumor, dysplasia, stroma and normal.

 7. Are there differences in the amount of proteins captured on each layer?

 Yes. The amount of protein eluted from the tissue section is gradually depleted during its transit through the layers, so the last layer receives less protein than the first. This is fully compensated for by a process of normalization of biomarker signals to total-protein signals that are measured in tandem on each layer. Please see the White Paper on this website for further information or contact us at MultiplexedIHC@2020gene.com .

 

Tissues and antibodies.

 1. Which antibodies can be used in L-IHC?

 Many different antibodies can be used in L-IHC, including those pre-qualified by their manufacturers for use in immunohistochemistry, as well as those simply pre-qualified for western blot use only. Our White Paper on this website contains a current list of the many antibodies which have been successfully used to date.

 2. How can I ascertain that my antibody panel and my tissue specimens will work well in L-IHC?

 As a service, 20/20 can perform a preliminary set-up experiment to qualify the selected panel of primary antibodies  for use in L-IHC with your tissue preparations (or similar surrogates for precious samples).

 3. What kinds of tissue preparations can be analyzed with L-IHC?

 Biomarkers can be analyzed in tissue that has been routinely formalin fixed, as well as tissues fixed in non-crosslinking fixatives such as ethanol, or from post-fixed sections prepared from snap-frozen specimens. 

 4. Do pre-analytical variables of tissue handling and fixation influence the analysis of biomarkers in L-IHC?

 Tissue preparation steps are the same for L-IHC as for IHC. Considerations of time to fixation, time in fixative and antigen unmasking/retrieval would affect both techniques similarly.

 5. Is there a tissue size limitation for L-IHC analysis?

 There is no tissue size limitation; L-IHC can used to analyze samples from surgical samples as well as small biopsies, core biopsies, and tissue microarrays.  This is especially important  wherein with routine IHC  there many not be enough tissue to analyze all of the biomarkers of interest.

Experiment design, data analysis and presentation of results

 

1. What type of results can best be obtained by using L-IHC?

 Limiting amounts of precious tissue can be analyzed for much larger numbers of analytes by using L-IHC. Ratio measurements between biomarkers will be a much more accessible metric and may provide new information of unique significance. For an example, please see the White Paper in this website.

 2. Where is the analysis performed and what software is used?

 The service includes data analysis using Image-Pro, ImageQuant and other software packages.

 3. What is the format of the data reported to the user of the service?

 The numerical data are presented in spreadsheet and/or table formats, and various graphic analyses can be tailored to the user’s specifications. The service includes an analysis of relevant statistics that can be determined from the assay metrics. Patient medical information should not be provided to the service. All reported data are for research use only, and are not meant to be used in the treatment of patients. 

Competitive Advantages

1. What are the advantages of L-IHC vs. RNA expression analysis?

L-IHC analyzes proteins that correspond directly to the tissue phenotype and therefore impact directly on the prognosis and are therapeutic targets.  It can be used to determine activation of specific signal transduction pathways through analysis of phosphorylated fraction to total protein for the different proteins along the pathway.  Many of the gene expression-based platforms analyze RNA derived from tissue that has been homogenized and therefore the relationship between histologic regions of interest and gene expression is not present.  There is the potential that RNA from contaminating cells may confound results.

 2. What are the advantages of L-IHC vs. protein analysis using mass spectrometry?

L-IHC is an antibody-based platform for analysis of biomarkers and therefore is readily determined.  Expression of many biomarkers can be correlated to a very small histologic region of interest on the tissue section that may be comprised of a few cells.  Mass spectrometry requires a significantly larger number of cells and may be of limited utility with tissue samples that have very small regions of interest.