![]() Designed for biologists, CellProfiler performs multiple sample analyses simultaneously, providing a high-throughput platform without the need to adjust the configuration or the user to possess programming skills. In the current investigation, we have employed the use of the freely downloadable open-source software CellProfiler 7.ĬellProfiler is a multipurpose software that has numerous advantages to already existing image analysis programs 7, 8. A large number of image analysis systems are currently available associated with robust staining platforms (reviewed by 2). Image analysis should support and not complicate the work of the pathologist. ![]() Therefore, within an image analysis system, identification of the chromogenic dye together with the nuclear stain is paramount. Most routine clinical pathology laboratories employ chromogenic detection methods, in particular 3,3′-diaminobenzidine (DAB) together with nuclear staining by haematoxylin. All of these factors can have a significant influence on the outcome. When establishing IHC image analysis routines, a number of factors need to be taken into consideration in order to develop robust reproducible procedures, in particular ensuring standardized sample preparation, the IHC protocol stages, and associated signal development 5. Therefore, computer-aided diagnostics offer significant advantages to pathologist working practices to provide reproducible, reliable, and accurate diagnostic assessments of tissue specimens and to move IHC from a qualitative/semiquantitative technique to more robust quantitative outputs 6. Many research reports still apply manual scoring as the golden standard to withdraw quantitative data from IHC, which makes inter-study comparisons difficult and requires validation by more than one observer to reduce variation in a time consuming practice 1, 4, 5. However, these nonstandardized evaluations remain a challenge even to an experienced pathologist and ultimately diagnostics may suffer from subjectivity, which may influence clinical outcome 4. In addition to stated descriptive terms, such as “band-like,” “heavy,” and “absent,” current pathological routines typically involve subjective visual manual assessment of IHC-stained specimens that are graded by percentage or via contrived groups such as −, +, ++, and +++ 4. Immunohistochemistry (IHC), as an aid to histopathological analyses, provides auxiliary information concerning spatial distribution and magnitude of cells or biomarkers within a tissue. With the movement toward personalized diagnostics and novel treatment options in the fields of cancer and inflammatory diseases, the use of digital pathology scanning systems with adjunctive image analysis tools has rapidly gained momentum in recent years 1- 3. CellProfiler allowed rapid high processing capacity of chromogenically stained chronic inflammatory tissue that was reliable, accurate, and reproducible and highlights potential applications in research diagnostics. CellProfiler demonstrated versatility with the ability to assess large numbers of images and allowed additional parameters to be quantified. Furthermore, CellProfiler allowed the determination of multiple variables simultaneously, such as area stained and masking to remove any nonstained tissue and white gaps, which also demonstrated reliable agreement ( r 2 = >0.9). Scatterplots and Bland–Altman plots demonstrated strong agreement between the manual counters and CellProfiler, with the number of positively stained cells robustly correlating ( r 2 = 0.938). Validation comparisons between the manual counters demonstrated strong intra-observer concordance ( r 2 = 0.979), particularly when cell numbers were less than 100. Inter-observer and inter-platform agreements were assessed by scatterplots with linear regression and Bland–Altman plots. ![]() Digitized images were manually counted and subjected to image analysis in CellProfiler. Oral mucosal biopsies from patients with chronic graft-versus-host disease were stained for CD4. The current investigation aimed to compare CellProfiler quantitative chromogenic IHC analyses against the gold standard manual counting. The open-source digital analysis software, CellProfiler has been extensively used for fluorescently stained cells/tissues however, chromogenic IHC staining is routinely used in both pathological and research diagnostics. ![]() Visual grading of chromogenically stained immunohistochemical (IHC) samples is subjective, time consuming, and predisposed to considerable inter- and intra-observer variations.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |