Semiquantitative analysis of immunohistochemical staining

The immunohistochemistry slides were independently examined by two observers. We utilized immunohistochemical scores (IHS) based on the German Immuno-Reactive score [24]. The IHS was calculated by combining an estimate of the percentage of immunereactive cells (quantitative score) with an estimate of the staining intensity (staining intensity score). No staining was scored as 0; staining of 1–10% as 1; 11–50% as 2; 51–80% as 3; and 81–100% as 4. The staining intensity was rated on a scale of 0 to 3, with 0 representing no staining, 1 weak staining, 2 moderate staining, and 3 strong staining. The raw data were converted to IHS by multiplying the quantity score and the intensity score. A moderate to strong level (HIS > 4) was considered to be positive, and weak or absent expression (IHS 0–4) was considered to be negative.