IHC scoring
H-score calculator
Enter the percentage of cells stained at each intensity, or the number of cells you counted, and get the H-score on its 0 to 300 scale together with the percent positive. Below are the formula, a worked example, what the number can and cannot tell you, and how image analysis software reaches the same score.
How the H-score is calculated
The H-score (histoscore) puts two things into one number: how many cells are stained and how strongly. Each cell of the population you are scoring, usually the tumour cells, is graded 0 (negative), 1+ (weak), 2+ (moderate) or 3+ (strong). The percentage of cells at each grade is multiplied by that grade and the three products are added.
H-score = 1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)
Worked example: 30 % of cells stain weakly, 20 % moderately and 10 % strongly; the other 40 % are negative. H-score = 30 × 1 + 20 × 2 + 10 × 3 = 100, with 60 % of cells positive. From counts it is the same sum: of 400 cells, 120 weak, 80 moderate and 40 strong give (120 + 160 + 120) ÷ 400 × 100 = 100.
The score was introduced for oestrogen receptor staining in the 1980s, as an evaluation that "incorporated both intensity and distribution of staining", and is now used for many nuclear, cytoplasmic and membrane markers.
What the number can and cannot tell you
- Different slides, same score: 100 % weak cells and 33 % strong cells both give about 100. Report the percent positive, or the percentage at each intensity, beside the H-score.
- No universal cut-off: the score that counts as "high" or "positive" depends on the marker, the antibody, the assay and the study. Take the threshold from the paper or guideline you are following, and state it.
- Intensity by eye is subjective: where weak ends and moderate begins differs between observers. Score against controls stained in the same run, and have a second reader score a sample of the slides.
- Score the right cells: count only the cell population the marker is read in, and leave out stroma, lymphocytes, necrosis and edge artefacts unless your protocol says otherwise.
- Count enough cells: a score from a few dozen cells moves a lot with each cell. Several hundred cells over representative fields is a common minimum.
Getting the same score from software
Image analysis software reaches these scores by the same arithmetic, with the eye replaced by a measurement. It finds every nucleus in the region, separates the brown DAB stain from the blue haematoxylin by colour deconvolution, measures the mean DAB optical density of each nucleus, and grades it against thresholds you set: one threshold for positive or negative, three for 1+, 2+ and 3+.
- QuPath (free, open source): Positive cell detection with one threshold gives percent positive; with three thresholds it also reports the H-score.
- Fiji / ImageJ (free): Colour Deconvolution with the H DAB vectors, then threshold and count; the scoring arithmetic is yours to do.
- SlideScope: counts nuclei in the region you choose, grades each one, and reports percent positive, H-score and Allred score together, using the same definitions as QuPath. You can also ask in plain language with Ask Your Slide; every number comes from the local tool, with outlines to check. Research use only.
Whichever you use, the thresholds are choices: write them in your methods, check the outlines against the image, and keep them fixed across the slides you compare. Step by step: how to quantify IHC staining.
Questions
How do you calculate an H-score?
Multiply the percentage of weakly stained cells by 1, moderately stained cells by 2 and strongly stained cells by 3, then add the three. With 30 % weak, 20 % moderate and 10 % strong the H-score is 30 + 40 + 30 = 100.
What is the range of the H-score?
0 to 300. A score of 0 means no cell is stained; 300 means every cell is strongly stained (100 % × 3).
What H-score is positive?
There is no universal cut-off. The threshold depends on the marker, the antibody and assay, and the study or guideline you follow, so take it from that source and report it with your result.
What is the difference between the H-score and the Allred score?
Both combine how many cells stain with how strongly. The H-score weights the percentage at each intensity and runs from 0 to 300. The Allred score adds a proportion score (0 to 5) to one average intensity score (0 to 3) and runs from 0 to 8.
Can software calculate the H-score?
Yes. QuPath reports it when you set three intensity thresholds in positive cell detection, and SlideScope reports percent positive, H-score and Allred score for a region using the same definitions. Both grade each nucleus by its mean DAB optical density, so the thresholds you choose belong in your methods.
Is this calculator free?
Yes. It runs in your browser and sends nothing anywhere. SlideScope, the desktop microscopy and pathology app made by the same team, is a paid app with a free sample library (every tool) and a subscription for your own images.
Sources
- McCarty KS Jr et al. Use of a monoclonal anti-estrogen receptor antibody in the immunohistochemical evaluation of human tumors. Cancer Res 1986;46(8 Suppl):4244s-4248s (the HSCORE)
- Wen Z et al. Deep learning-based H-score quantification of immunohistochemistry-stained images. Mod Pathol 2023 (formula and 0 to 300 range)
- QuPath documentation: cell classification, intensity thresholds and H-score
These calculators do arithmetic for research and teaching. They are not a diagnostic tool: scoring for patient care follows the validated assay, the current guideline for that marker and a pathologist's judgement.
Score the slide instead of counting by hand
SlideScope counts the nuclei in a region of your slide, grades each one by its DAB staining, and reports percent positive, H-score and Allred score with the outlines to check, on your own computer. Or type the question: Ask Your Slide runs the same tools and shows the evidence. Research use only. Try every tool free on the built-in samples; opening your own images needs a subscription. Already decided? Subscribe now.