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1.  Subcellular Localization of p27 and Prostate Cancer Recurrence: Automated Digital Microscopy Analysis of Tissue Microarrays 
Human pathology  2011;42(6):873-881.
Previous investigations have linked decreased nuclear expression of the cell cycle inhibitor p27 with poor outcome in prostate cancer. However, these reports are inconsistent regarding the magnitude of that association and its independence from other predictors. Moreover, cytoplasmic translocation of p27 has been proposed as a negative prognostic sign. Given the cost and accuracy limitations of manual scoring, particularly of tissue microarrays (TMAs), we determined if laser-based fluorescence microscopy could provide automated analysis of p27 in both nuclear and cytoplasmic locations, and thus clarify its significance as a prognostic biomarker.
We constructed TMAs covering 202 recurrent cases (rising PSA) and 202 matched controls without recurrence. Quadruplicate tumor samples encompassed 5 slides and 1,616 cancer histospots. Cases and controls matched on age, Gleason grade, stage and hospital. We immunolabeled epithelial cytoplasm with Alexa647®; p27 with Alexa488®; and nuclei with DAPI. Slides were scanned on an iCys® laser scanning cytometer. Nuclear crowding required a stereological approach - random arrays of circles (phantoms) were layered on images and the content of each phantom analyzed in scatterplots.
Both nuclear and cytoplasmic p27 were significantly lower in cases vs. controls (P=0.014, P=0.004, respectively). Regression models controlling for matching variables plus PSA showed strong linear trends for increased risk of recurrence with lower p27 in both nucleus and cytoplasm (highest vs lowest quartile, OR=0.35, P=0.006). Manual scoring identified an inverse association between p27 expression and tumor grade, but no independent association with recurrence.
In conclusion, we developed an automated method for subcellular scoring of p27 without the need to segment individual cells. Our method identified a strong relationship, independent of tumor grade, stage and PSA, between p27 expression – regardless of subcellular location - and prostate cancer recurrence. This relationship was not observed with manual scoring.
PMCID: PMC3095701  PMID: 21292307
prostate cancer; prognosis; p27Kip1; automated image analysis; tissue microarray
2.  Laser scanning cytometry for automation of the micronucleus assay 
Mutagenesis  2011;26(1):153-161.
Laser scanning cytometry (LSC) provides a novel approach for automated scoring of micronuclei (MN) in different types of mammalian cells, serving as a biomarker of genotoxicity and mutagenicity. In this review, we discuss the advances to date in measuring MN in cell lines, buccal cells and erythrocytes, describe the advantages and outline potential challenges of this distinctive approach of analysis of nuclear anomalies. The use of multiple laser wavelengths in LSC and the high dynamic range of fluorescence and absorption detection allow simultaneous measurement of multiple cellular and nuclear features such as cytoplasmic area, nuclear area, DNA content and density of nuclei and MN, protein content and density of cytoplasm as well as other features using molecular probes. This high-content analysis approach allows the cells of interest to be identified (e.g. binucleated cells in cytokinesis-blocked cultures) and MN scored specifically in them. MN assays in cell lines (e.g. the CHO cell MN assay) using LSC are increasingly used in routine toxicology screening. More high-content MN assays and the expansion of MN analysis by LSC to other models (i.e. exfoliated cells, dermal cell models, etc.) hold great promise for robust and exciting developments in MN assay automation as a high-content high-throughput analysis procedure.
PMCID: PMC3107611  PMID: 21164197
3.  Experimental Validation Of Peptide Immunohistochemistry Controls 
Peptide immunohistochemistry (IHC) controls are a new quality control format for verifying proper IHC assay performance, offering advantages in high throughput automated manufacture and standardization. We previously demonstrated that formalin-fixed peptide epitopes, covalently attached to glass microscope slides, behaved (immunochemically) in a similar fashion to the native protein in tissue sections. To convert this promising idea into a practical clinical laboratory quality control tool, we tested the hypothesis that the quality assurance information provided by peptide IHC controls accurately reflects IHC staining performance amongst a diverse group of clinical laboratories. To test the hypothesis, we first designed and built an instrument for reproducibly printing the controls on microscope slides and a simple software program to measure the color intensity of stained controls. Automated printing of peptide spots was reproducible, with CVs of 4−8%. Moreover, the peptide controls were stable at ≤4° C for at least seven months, the longest time duration we tested. A national study of 109 participating clinical laboratories demonstrated a good correlation between a laboratory's ability to properly stain formalin-fixed peptide controls to their ability in properly staining a 3+ HER-2 formalin-fixed tissue section mounted on the same slide (r = 0.87). Therefore, peptide IHC controls accurately reflect the analytical component of an IHC stain, including antigen retrieval. Besides its use in proficiency survey testing, we also demonstrate the feasibility of applying peptide IHC controls for verifying intra-laboratory IHC staining consistency, using Levy-Jennings charting.
PMCID: PMC2672113  PMID: 19077907
Immunohistochemistry; Controls; Standardization; Peptide; HER-2; Quality Control

Results 1-3 (3)