The H score in RNAscope is a widely used semi-quantitative scoring method that provides a comprehensive measure of target RNA expression levels within tissue samples. It consolidates both the intensity of the signal (number of RNA dots per cell) and the percentage of cells showing a particular signal intensity, offering a robust and reproducible way to assess gene expression.
This score is assigned based on the number of cells exhibiting specific ranges of dots per cell. Such an analysis can be effectively performed manually by a trained pathologist or a skilled scientist, ensuring accurate interpretation of RNA expression patterns.
Understanding the RNAscope H Score
RNAscope technology allows for the in situ visualization and quantification of individual RNA molecules as discrete dots within cells. While simply counting dots per cell provides quantitative data, the H score provides a consolidated value that reflects the overall expression profile across a tissue section.
The H score typically ranges from 0 to 300 and is calculated using the following formula:
H-score = (1 × % cells with 1 dot) + (2 × % cells with 2-3 dots) + (3 × % cells with 4-9 dots) + (4 × % cells with ≥10 dots/clumps)
Note: While some sources may use slightly different dot count ranges or intensity multipliers (e.g., 0-3 scale for intensity), the underlying principle of weighting cell percentages by signal intensity remains consistent. For RNAscope, the common interpretation is based on a 0-4 scale for dot count per cell categories.
Scoring Categories and Interpretation
To derive the H score, cells are categorized based on the number of RNA dots observed per cell. These categories reflect different levels of RNA expression:
| Scoring Category | Dots Per Cell Description | Intensity Multiplier |
|---|---|---|
| 0 | No dots, or <1 dot per cell | 0 |
| 1 | 1-3 dots per cell (low expression) | 1 |
| 2 | 4-9 dots per cell (medium expression) | 2 |
| 3 | 10-15 dots per cell, small clusters | 3 |
| 4 | >15 dots per cell, larger clusters/clumps (high expression) | 4 |
- Process: For each tissue area of interest, a trained observer estimates the percentage of cells falling into each of these categories. These percentages are then multiplied by their respective intensity multipliers (0-4) and summed to yield the final H score.
- Example: If 50% of cells have 1-3 dots (Intensity 1), 30% have 4-9 dots (Intensity 2), and 20% have ≥10 dots (Intensity 3), the H score might be calculated as (1*50) + (2*30) + (3*20) = 50 + 60 + 60 = 170. (This uses a slightly simplified version of the formula, showing how percentages of cells at different levels contribute). For the standard RNAscope 0-4 scale, a more precise example:
- 10% cells with 1-3 dots (level 1)
- 40% cells with 4-9 dots (level 2)
- 30% cells with 10-15 dots (level 3)
- 20% cells with >15 dots/clumps (level 4)
- H-score = (1 10) + (2 40) + (3 30) + (4 20) = 10 + 80 + 90 + 80 = 260
Advantages of Using the H Score
- Comprehensive Assessment: Unlike simple dot counting, the H score accounts for the heterogeneity of expression within a cell population, providing a more holistic view.
- Semi-Quantitative Nature: It bridges the gap between purely qualitative visual assessment and highly quantitative, but often more complex, image analysis software.
- Reproducibility: When performed by trained individuals, the H score offers a reproducible method for comparing RNA expression levels across different samples or experimental conditions.
- Clinical Relevance: The H score methodology, originally used in immunohistochemistry (IHC) for protein expression, has been successfully adapted for RNAscope, making it valuable for research and potential clinical applications where a standardized scoring system is beneficial.
For more information on RNAscope technology and its applications, you can refer to resources from Advanced Cell Diagnostics (ACD) Bio-Techne, the developer of RNAscope.
