Monoclonality analysis

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Monoclonality analysis Service

In the era of precision medicine, monoclonality analysis has become a core tool for hematologic malignancy diagnosis, therapeutic efficacy monitoring, and immunology research. With the maturity of next-generation sequencing (NGS) technology, NGS-based monoclonality analysis services are gradually replacing traditional methods. With their unprecedented sensitivity, quantitative capabilities, and high throughput, they provide stronger technical support for clinical and scientific research. This article will provide an in-depth analysis of the working principles, standardized workflow, and wide-ranging application scenarios of NGS-based monoclonality analysis.

Service Principle

A Paradigm Revolution from "Size Analysis" to "Sequence Analysis"
Traditional PCR-capillary electrophoresis technology can only indirectly infer clonality based on the size (length) of the amplified product. NGS, however, directly reads the sequence of each DNA molecule, achieving a leap from "macroscopic" to "microscopic" analysis.

ts core principle remains based on the biology of lymphocyte-specific gene rearrangement:
* During V(D)J rearrangement, immunoglobulin (Ig) genes (such as IGH, IGK, and IGL) in B cells and T cell receptor (TCR) genes (such as TCRB and TCRG) in T cells generate extremely diverse complementarity-determining region 3 (CDR3) sequences.

* Polyclonal populations exhibit a high diversity of CDR3 sequences, with no single sequence dominating.

* Monoclonal or oligoclonal populations exhibit a significant preponderance of one or a few identical CDR3 sequences, resulting in a significant dominance in sequence reads and frequency.

Next-generation sequencing (NGS) technology uses high-throughput sequencing of the target region (typically the CDR3 region) after PCR amplification, simultaneously generating hundreds of thousands or even millions of sequence reads. Bioinformatics analysis accurately calculates the frequency of each unique CDR3 sequence, enabling not only qualitative assessment of clonality but also quantitative assessment of the proportion of dominant clones, allowing the tracking of even extremely small clonal populations.

Standardized Workflow

Our NGS-based monoclonality analysis service adheres to a rigorous, standardized set of procedures to ensure data accuracy and reproducibility.

Sample Preparation and DNA Extraction

This service can process a variety of sample types, including peripheral blood, bone marrow, formalin-fixed paraffin-embedded (FFPE) tissue, and fine-needle aspirates. Extracting high-quality, intact genomic DNA is essential for success. Degraded samples, such as FFPE, require specialized extraction and library construction techniques.

Multiplex PCR Amplification and Library Construction

This is a critical step in the process. A carefully designed multiplex primer panel is used to simultaneously amplify all common V and J segments of the target gene (e.g., IGH V-J rearrangements). This multiplex PCR system ensures high coverage and sensitivity, effectively capturing the vast majority of clones. Subsequently, the amplified products are ligated with sequencing adapters and sample indexes (barcodes) to construct NGS libraries for sequencing

High-throughput Sequencing

The constructed library is subjected to high-throughput sequencing on an NGS platform such as Illumina or Ion Torrent. Paired-end sequencing is typically used to ensure coverage of the entire CDR3 region and obtain accurate sequence information.

Bioinformatics Analysis

This is the "brain" of NGS technology and its core value. The analysis process typically includes:
* Raw data quality control and filtering: Removal of low-quality reads and adapter sequences.

* Sequence alignment and annotation: Reads are aligned with a reference gene database to accurately identify V, D, and J segments and determine unique CDR3 nucleotide and amino acid sequences.

* Clonality assessment and quantification: The frequency of each unique CDR3 sequence is counted. By setting a threshold (e.g., a dominant clone exceeding a certain percentage of total reads), a sample is scientifically determined to be monoclonal, polyclonal, or oligoclonal.

* Clonal evolution tracking: For minimal residual disease (MRD) monitoring, precise alignment with tumor-specific clone sequences detected at initial diagnosis enables ultra-sensitive quantitative tracking.

Report Generation and Interpretation

The service provider will provide a detailed, easy-to-understand report, including clonality conclusions, sequence information of the dominant clone, frequency quantification results, clonal diversity indicators (such as the Shannon index), and more, accompanied by professional clinical or scientific interpretation.

Application Scenarios

The introduction of NGS technology has greatly expanded the depth and breadth of applications for monoclonality analysis.

Accurate Diagnosis and Typing of Hematologic Malignancies:
In the diagnosis of diseases such as lymphoma and leukemia, the sequence-level evidence provided by NGS is more convincing than traditional electrophoresis, providing crucial diagnostic evidence, especially in difficult cases.

The "Gold Standard" for Minimal Residual Disease (MRD) Monitoring:
This is the most valuable application of NGS monoclonality analysis. Its sensitivity can reach as high as 10^-6 (detecting one tumor cell in one million normal cells), far exceeding flow cytometry and traditional PCR. By regularly monitoring changes in the frequency of tumor-specific clones within a patient, it can predict relapse risk, assess treatment response, and guide personalized treatment decisions. It has become a vital tool in clinical trials and daily care.

Immune Repertoire Research:NGS can comprehensively characterize the diversity, clonal structure, and dynamic changes of the T cell and B cell receptor repertoires. This is of irreplaceable value in basic and translational research on autoimmune diseases, infectious diseases, immunosenescence, and the tumor immune microenvironment.

Cell Therapy Product Quality Control:During the production of cell therapy products such as CAR-T, it is imperative to confirm that the final product is a functional T cell clone, not a nonspecifically expanded clone. NGS analysis can verify the product's polyclonality and diversity, ensuring its safety and efficacy.

Dynamic Study of Therapeutic Response and Clonal Evolution:
Longitudinal sampling and sequencing can reveal the clonal evolution of tumors under the pressure of chemotherapy, targeted therapy, or immunotherapy, providing insights into drug resistance mechanisms and the development of new combination therapy strategies.

NGS-based monoclonality analysis services represent the pinnacle of technology in this field, elevating the previously "black and white" clonality assessment to a new level of precision that allows for "quantitative, dynamic, and traceable" analysis. With the continuous decline in sequencing costs and the increasing intelligence of bioinformatics tools, this service is moving from large research centers to routine clinical laboratories. It is bound to become the core engine driving the development of precision hematology and immunology, bringing unprecedented in-depth insights to more patients and researchers.

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Email: sales@cobioer.com

Service Principle

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