Determination of HMGB1 and CD68 expression in tissue microarrays. (A) Representative samples of HMGB1 expression in (i-ii) reactive and (iii-vi) CLL lymph nodes. Images were taken with a Leixa DM2500 microscope: i, iii, and v, original magnification ×200); ii, iv, and vi, original magnification ×400. (B) Overall HMGB1 expression. HMGB1 expression was determined by a computerized image analysis Ariol system using pathologist-trained visual parameters. All cores were reviewed manually before scoring, and only intact cores were used for scoring. Each datum represents an average of triplicate cores. There was a statistically significant decrease in expression of percentage of cells expressing HMGB1 in CLL compared with reactive LNs. (C) Cytoplasmic HMGB1 expression. Numbers of cytoplasmic HMGB1-positive cells were counted blindly by 2 reviewers in 4 high power fields (hpfs) and expressed as a mean value from triplicate cores. (D) Overall survival of CLL patients based on low (<50) and high (>50) numbers of cells containing cytoplasmic HMGB1 in 4 counted hpfs, with the cutoff determined using X-tiles software. (E) Expression of CD68 in (i-ii) reactive and CLL-LN with (iiii) lower and (iv) higher CD68 expression. (F) Statistical comparison of CD68 expression in CLL-LN and RA-LN. (G) Overall survival of CLL patients based on low (<10%) and high (>10%) expression of CD68.