Pixelwise H-score: A novel digital image analysis-based metric to quantify membrane biomarker expression from immunohistochemistry images.

Immunohistochemistry (IHC) assays play a central role in evaluating biomarker expression in tissue sections for diagnostic and research applications. Manual scoring of IHC images, which is the current standard of practice, is known to have several shortcomings in terms of reproducibility and scalability to large scale studies. Here, by using a digital image analysis-based approach, we introduce a new metric called the pixelwise H-score (pix H-score) that quantifies biomarker expression from whole-slide scanned IHC images. The pix H-score is an unsupervised algorithm that only requires the specification of intensity thresholds for the biomarker and the nuclear-counterstain channels. We present the detailed implementation of the pix H-score in two different whole-slide image analysis software packages Visiopharm and HALO. We consider three biomarkers P-cadherin, PD-L1, and 5T4, and show how the pix H-score exhibits tight concordance to multiple orthogonal measurements of biomarker abundance such as the biomarker mRNA transcript and the pathologist H-score. We also compare the pix H-score to existing automated image analysis algorithms and demonstrate that the pix H-score provides either comparable or significantly better performance over these methodologies. We also present results of an empirical resampling approach to assess the performance of the pix H-score in estimating biomarker abundance from select regions within the tumor tissue relative to the whole tumor resection. We anticipate that the new metric will be broadly applicable to quantify biomarker expression from a wide variety of IHC images. Moreover, these results underscore the benefit of digital image analysis-based approaches which offer an objective, reproducible, and highly scalable strategy to quantitatively analyze IHC images.

Immunocytochemical and in situ hybridization studies of gastrin after cisplatin treatment.

Cisplatin treatment (9 mg/kg) causes bloating of the stomach, an increase in gastric acid, and ulceration in rats. Gastrin, a gut peptide, plays an important role in regulating gastric acid production. To study the role of gastrin in this increased gastric acid production after cisplatin treatment, male Wistar rats (100-150 g) were treated with cisplatin (9 mg/kg) in five divided doses over 5 consecutive days. The rats were sacrificed 1, 6, 10, or 15 days after the last treatment. As measured by immunocytochemistry, in situ hybridization, Northern blot, and dot-blot techniques, gastrin was found to be below detectable limits just 1 day after cisplatin treatment. However, 10-15 days after the last injection, the levels for both gastrin and its mRNA gradually recovered to normal. Northern blot studies showed that decreased somatostatin mRNA parallels the changes of gastrin and its mRNA. These results suggest that after cisplatin treatment the increased gastric acid production in rat stomach is independent of gastrin. This decrease of gastrin production is not under the influence of somatostatin, which also decreased after cisplatin treatment.

Semen quality characteristics of dairy goats.

Semen was collected, processed, and frozen from five dairy bucks for 2 successive yr for use in quality classification and evaluation for inclusion in artificial insemination programs. Semen was evaluated for volume, initial, postthaw and 37 degrees C incubated percent progressive motility, percent postthaw 3-h 37 degrees C incubated intact acrosomes, autoagglutination, whey-induced agglutination, and percent primary, secondary, and tertiary abnormalities. Significant high correlations were found between: percent progressive motility and percent intact acrosomes, percent intact acrosomes and percent autoagglutination, and percent autoagglutination and percent whey agglutination. Means of the postthaw quality parameters, percent progressive motility, percent intact acrosomes, and percent primary and secondary abnormalities were used to categorize ejaculates within each incubation time (0 and 2 h). At 0 h, 25 ejaculates were classified as high quality and 11 were low quality. Using 2-h data, 19 ejaculates were classified as high quality and 17 as low. Inclusion of both agglutination parameters in the 2-h data analysis resulted in 13 ejaculates categorized as high and 23 as low quality. Based on assessment with techniques used in bovine artificial insemination programs, semen quality of goat semen could be used to discriminate between acceptable or unacceptable ejaculates. Based on recommended sperm numbers per inseminate and average ejaculate characteristics the low number of marketable units per ejaculate would make incorporation of goats into existing artificial insemination programs prohibitive.