Do the spot diameter and replicate spots of custom tissue microarrays have a significant impact on immunohistochemistry quantification?

For immunohistochemistry quantification and H-score, I generally recommend prioritizing 1.5 mm. Although 1.0 mm offers higher throughput and allows more spots per slide, the smaller tissue area makes edge effects and local heterogeneity more pronounced in quantification, which can easily lead to H-score fluctuation. A single 1.5 mm spot allows more cells to be observed, resulting in more robust counting, especially for tumors with strong heterogeneity such as lung cancer and gastric cancer. If higher throughput is absolutely necessary, 1.0 mm can be considered with additional replicate spots and cases, but for quantification, 1.5 mm is the safer choice.

Edge effects are not necessarily caused by spot diameter; they are more related to sectioning, baking, antigen retrieval, and antibody coverage. If manufacturers standardize the use of the same paraffin, unify section thickness, avoid edge spots in quantification, and use humid chambers and sufficient antibody, edge effects will be significantly reduced. For batch-to-batch consistency, suppliers should be required to assign fixed pathology technicians, use the same model of microtome and the same lot of reagents, and stain each batch of chips with the same reference sample. You can place several positive/negative control spots on each chip to correct for batch-to-batch drift. The contract should specify the acceptable range for the coefficient of variation of H-score between batches.

You can ask the manufacturer to provide digital scans, but be sure to clarify the scan resolution, format, and whether export is by whole slide or by spot. H-score analysis is best performed under the same scan parameters in a single session, as brightness differences between scans of different batches can affect quantification. Request the original brightfield images and the H-score calculation parameters or scripts so that you can review them yourselves. If chips from multiple batches are to be compared, try not to mix different scanners. Analysis services can serve as one component of quality control, but the final statistics should ideally be reviewed by you or a pathologist for the key targets.

It is recommended to have at least 2 replicate spots, preferably 2–3, and the two replicate spots should not always be placed side by side in the same area of the chip; they should be distributed across different rows or different areas as much as possible to reduce systematic bias caused by local repair and uneven antibody incubation. If tumor heterogeneity is high or sample size permits, 3 spots per case can be used, preferentially taken from the region between the tumor center and the margin. Replicate spots are not simple duplicates; they should preferably come from different wax blocks or from areas at different depths of the same wax block, so that they can better represent the tumor as a whole.