MSI Testing Methods Compared: IHC, PCR-CE and NGS for Detecting Microsatellite Instability

Microsatellite instability, commonly known as MSI, is an important molecular biomarker in oncology. It is closely associated with defects in the DNA mismatch repair system, also known as dMMR. MSI status is widely used in cancer diagnostics, Lynch syndrome screening, prognosis evaluation and treatment decision-making.

As MSI testing becomes increasingly important in molecular pathology and precision oncology, laboratories need to understand the differences between available testing methods. The most commonly used approaches include immunohistochemistry, PCR combined with capillary electrophoresis, and next-generation sequencing.

Each method has its own advantages, limitations and ideal application scenarios. This article compares IHC, PCR-CE and NGS for MSI detection and explains why validated MSI reference standards are essential for reliable assay development and quality control.

Why MSI Testing Matters

MSI occurs when short repetitive DNA sequences, called microsatellites, become unstable due to insertion or deletion errors during DNA replication. In normal cells, these errors are corrected by the mismatch repair system. When mismatch repair function is defective, errors can accumulate, leading to changes in microsatellite length.

MSI testing is important because MSI/dMMR status can provide valuable information for:

• Screening for Lynch syndrome

• Characterizing tumor biology

• Supporting prognosis evaluation

• Guiding treatment-related decisions

• Developing and validating molecular diagnostic assays

• Supporting oncology research and biomarker studies

Because MSI status can influence clinical and research interpretation, the testing workflow must be accurate, reproducible and well validated.

Main MSI Testing Methods

The three major approaches used for MSI or dMMR assessment are:

1. Immunohistochemistry, or IHC

2. PCR-based MSI testing, especially PCR-CE

3. Next-generation sequencing, or NGS

Although these methods are related, they do not measure exactly the same thing. IHC evaluates the expression of mismatch repair proteins, while PCR-CE and NGS directly or computationally assess microsatellite instability at the DNA sequence level.

IHC for MMR Protein Expression

Immunohistochemistry is one of the most widely used methods for assessing mismatch repair deficiency. Instead of directly detecting microsatellite length changes, IHC evaluates the expression of key MMR proteins in tumor tissue.

The commonly tested proteins include:

• MLH1

• MSH2

• MSH6

• PMS2

If one or more of these proteins are absent, the tumor may be considered mismatch repair deficient. Because dMMR is strongly associated with MSI-H, IHC is often used as a practical screening method.

Advantages of IHC

IHC is widely available in pathology laboratories and can be integrated into routine tissue-based workflows. It provides visual information about protein expression and can help identify which MMR protein may be lost.

For example, loss of MLH1 and PMS2 expression may suggest a different biological pattern than isolated loss of MSH6. This makes IHC useful not only for screening but also for guiding further investigation.

Limitations of IHC

IHC does not directly measure microsatellite instability. It only evaluates protein expression. In some cases, protein expression may be retained even when functional defects are present. Interpretation may also be affected by tissue quality, staining conditions, fixation, antibody performance and pathologist experience.

Therefore, IHC is useful but may need to be supported by molecular testing in certain situations.

PCR-CE for Microsatellite Marker Analysis

PCR-CE is a molecular method that directly analyzes selected microsatellite markers. In this workflow, specific microsatellite regions are amplified by PCR and then analyzed using capillary electrophoresis.

The purpose is to detect changes in fragment length. Tumor DNA is often compared with matched normal DNA to determine whether microsatellite markers have shifted in size. If multiple markers show instability, the sample may be classified as MSI-H.

Common MSI markers include mononucleotide markers such as BAT-25, BAT-26, NR-21, NR-24 and MONO-27. Some systems may also include additional markers such as NR-27 or pentanucleotide markers for sample identification and quality control.

Advantages of PCR-CE

PCR-CE is a well-established method for MSI detection. It provides direct fragment size information for selected microsatellite loci and is suitable for laboratories that need a targeted and focused MSI workflow.

PCR-CE can be especially useful when laboratories want a dedicated MSI testing method rather than a broad genomic profiling approach.

Limitations of PCR-CE

PCR-CE usually analyzes a limited number of markers. Its performance depends on the selected marker panel, DNA quality, tumor content and interpretation criteria.

Another limitation is that PCR-CE may require matched normal DNA for more accurate comparison, depending on the assay design. Some workflows can be affected by sample quality or low tumor fraction.

Despite these limitations, PCR-CE remains an important method for MSI testing because of its targeted nature and clear fragment analysis output.

NGS-Based MSI Detection

Next-generation sequencing has become an increasingly important approach for MSI detection. NGS can assess many genomic regions simultaneously and can often be integrated into broader tumor profiling workflows.

MSI status may be inferred from:

• Targeted gene panels

• Whole-exome sequencing

• Whole-genome sequencing

• Other sequencing-based tumor profiling datasets

NGS-based MSI detection typically relies on bioinformatics algorithms that analyze microsatellite regions across sequencing data. Instead of examining only a few markers, NGS can evaluate a larger number of loci depending on the panel design and analytical pipeline.

Advantages of NGS

The biggest advantage of NGS is its ability to combine MSI detection with broader genomic profiling. A single NGS workflow can potentially provide information about mutations, copy number changes, tumor mutation burden, gene fusions and MSI status, depending on the assay design.

This makes NGS attractive for laboratories that already perform comprehensive cancer genomic testing.

NGS can also offer high-throughput analysis and may reduce the need for multiple separate assays when a validated sequencing workflow is already in place.

Limitations of NGS

NGS-based MSI detection requires careful validation. The result depends not only on wet-lab performance but also on sequencing depth, panel design, microsatellite loci coverage, tumor purity, DNA quality and bioinformatics algorithms.

Different NGS panels may not perform equally for MSI detection. A panel designed mainly for mutation detection may not automatically be optimal for MSI calling unless MSI performance has been specifically validated.

Therefore, reliable reference standards are especially important for NGS-based MSI assay development and pipeline verification.

IHC vs PCR-CE vs NGS: Key Differences

How to Choose the Right MSI Testing Method

There is no single MSI testing method that is ideal for every laboratory or every application. The best choice depends on the testing purpose, available sample type, laboratory infrastructure and validation requirements.

When IHC May Be Preferred

IHC may be preferred when a laboratory needs a tissue-based screening method for mismatch repair protein expression. It is commonly used in pathology workflows and can provide useful information about which MMR proteins are lost.

When PCR-CE May Be Preferred

PCR-CE may be preferred when a laboratory needs a focused molecular MSI assay with direct fragment size analysis. It is useful for dedicated MSI testing and for laboratories that want a targeted workflow based on known microsatellite markers.

When NGS May Be Preferred

NGS may be preferred when MSI detection is part of a broader cancer genomic profiling workflow. It is especially useful for laboratories that already use targeted panels or comprehensive sequencing assays.

However, NGS-based MSI detection should be validated with appropriate reference materials to ensure that the assay and bioinformatics pipeline can accurately classify MSI-H and MSS samples.

Why MSI Reference Standards Are Important Across Platforms

Regardless of the method used, MSI testing requires reliable validation and quality control. Reference standards help laboratories confirm that their assays can correctly detect known MSI status.

MSI reference standards can support:

• Assay development

• Analytical validation

• Limit of detection evaluation

• Positive and negative control testing

• Inter-run reproducibility studies

• Platform comparison

• Operator training

• Routine quality control

• Bioinformatics pipeline verification for NGS

For PCR-CE, reference standards can help verify marker amplification, fragment sizing and MSI classification.

For NGS, reference standards can help evaluate sequencing performance, panel coverage, MSI calling algorithms and overall workflow consistency.

For IVD developers, reference standards can support product development and performance evaluation across different assay designs.

CB-Gene MSI Reference Standards for PCR-CE and NGS Workflows

CB-Gene provides MSI Reference Standards designed to support molecular diagnostic assay validation and quality control.

The CB-Gene MSI Reference Standard product includes paired genomic DNA samples, including tumor-derived DNA and matched or reference normal DNA. The product set includes both microsatellite stable samples and MSI-H samples, making it useful for evaluating whether an assay can correctly distinguish MSS from MSI-H.

According to CB-Gene product information, the reference standards are confirmed by PCR-CE assay and NGS assay. The PCR-CE MSI analysis system includes multiple nucleotide markers such as BAT-25, BAT-26, MONO-27, NR-21, NR-24 and NR-27 for MSI typing.

This makes the standards suitable for laboratories developing, validating or monitoring MSI workflows based on PCR-CE or NGS platforms.

Applications of MSI Reference Standards

CB-Gene MSI Reference Standards can be used in several technical and development scenarios.

PCR-CE Assay Validation

Laboratories can use MSI-H and MSS standards to verify PCR amplification, capillary electrophoresis performance, marker stability and classification accuracy.

NGS Panel Validation

Sequencing laboratories can use reference standards to evaluate whether their NGS panel and analysis pipeline can correctly identify MSI-H and MSS status.

Bioinformatics Pipeline Verification

For NGS-based MSI detection, the computational pipeline is a critical part of the testing workflow. Reference standards with confirmed MSI status can help verify MSI calling algorithms and classification thresholds.

Quality Control

Reference standards can be included in routine quality control workflows to monitor assay consistency over time.

IVD Product Development

Diagnostic assay developers can use MSI reference materials during kit development, optimization and performance evaluation.

Conclusion

IHC, PCR-CE and NGS are all important methods for assessing MSI or mismatch repair deficiency, but they differ in what they measure and how they are used.

IHC evaluates MMR protein expression. PCR-CE directly analyzes selected microsatellite marker length changes. NGS enables MSI detection within broader cancer genomic profiling workflows.

Because each method has different strengths and limitations, laboratories should choose the appropriate workflow based on their testing goals, available infrastructure, sample type and validation needs.

Reliable MSI reference standards are essential for building confidence in assay performance. CB-Gene MSI Reference Standards provide confirmed MSI-H and MSS genomic DNA materials for PCR-CE and NGS-based MSI assay validation, helping laboratories and diagnostic developers improve consistency, reproducibility and confidence in MSI testing.

For more information about CB-Gene MSI Reference Standards, please contact CB-Gene for product details, technical specifications and application support.