Details

Name
DAB Stain Analysis
IQbot ID
IQBOT-0004
Version
1.0.0
Research Area(s)
Angiogenesis, Arthritis & Osteoarthritis, Cancer Research, Cardiovascular, Dermatology, Diabetes, Drug Toxicity & Metabolism, Genetics, Infectious Disease & Bacterial Adhesion, Neurology, Ophthalmology, Orthopedics, Reproductivity, Common Tools
Processing Time Expectation
Less than 2 Minutes per Image
Acquisition Hardware
Wide-Field, Optical Microscope in Bright-Field (Transmission) Mode Using a Color Mosaic CCD or Monochrome CCD with RGB Filter
Techniques, Dyes, Stains, & Proteins
3,3'-Diaminobenzidine (DAB), Horesradish Peroxidase (HRP), Hematoxylin Counterstain, Immunohistochemistry, Chromogen, Brown Oxidation Reaction
Sample Context
In Tissue
References
1.   Saunthararajah Y, Sekeres M, Advani A, Mahfouz R, Durkin L, Radivoyevitch T, Englehaupt R, Juersivich J, Cooper K, Husseinzadeh H, Przychodzen B, Rump M, Hobson S, Earl M, Sobecks R, Dean R, Reu F, Tiu R, Hamilton B, Copelan E, Lichtin A, Hsi E, Kalaycio M, Maciejewski J. Evaluation of noncytotoxic DNMT1-depleting therapy in patients with myelodysplastic syndromes. J Clin Invest. 2015 Mar 2;125(3):1043-55

 

Name
DAB Stain Analysis
IQbot ID
IQBOT-0004
Version
1.0.0
Research Area(s)
Angiogenesis, Arthritis & Osteoarthritis, Cancer Research, Cardiovascular, Dermatology, Diabetes, Drug Toxicity & Metabolism, Genetics, Infectious Disease & Bacterial Adhesion, Neurology, Ophthalmology, Orthopedics, Reproductivity, Common Tools
Processing Time Expectation
Less than 2 Minutes per Image
Acquisition Hardware
Wide-Field, Optical Microscope in Bright-Field (Transmission) Mode Using a Color Mosaic CCD or Monochrome CCD with RGB Filter
Techniques, Dyes, Stains, & Proteins
3,3'-Diaminobenzidine (DAB), Horesradish Peroxidase (HRP), Hematoxylin Counterstain, Immunohistochemistry, Chromogen, Brown Oxidation Reaction
Sample Context
In Tissue
References
1.   Saunthararajah Y, Sekeres M, Advani A, Mahfouz R, Durkin L, Radivoyevitch T, Englehaupt R, Juersivich J, Cooper K, Husseinzadeh H, Przychodzen B, Rump M, Hobson S, Earl M, Sobecks R, Dean R, Reu F, Tiu R, Hamilton B, Copelan E, Lichtin A, Hsi E, Kalaycio M, Maciejewski J. Evaluation of noncytotoxic DNMT1-depleting therapy in patients with myelodysplastic syndromes. J Clin Invest. 2015 Mar 2;125(3):1043-55

Output Parameters

Total Tissue Area (pixels)
The total area of tissue within the image.
Positive Nuclei or Region Count
The number of DAB positive regions within the image, independent of size.
Positive Nuclei or Region Area (pixels)
The total area of DAB staining across the entire image.

 

This section will identify any output parameters that are only output by the IQbot in its advanced configuration.

 

Total Tissue Area (pixels)
The total area of tissue within the image.
Positive Nuclei or Region Count
The number of DAB positive regions within the image, independent of size.
Positive Nuclei or Region Area (pixels)
The total area of DAB staining across the entire image.

Image Acquisition


This IQbot was developed to analyze images acquired using a wide-field, optical microscope in bright-field (transmission) mode using a color mosaic CCD or monochrome CCD with RGB filter. For accurate analysis of tissue area, sections must be counterstained with hematoxylin.

 
 

The DAB Stain Analysis IQbot has been built to accommodate the following imaging types only.

 24-bit RGB
 .tif
  .tiff
  .bmp
 .jpg
.jpeg
 
 

As we reference for IQbots in general here, the DAB Stain Analysis is only ultimately effective if you upload high quality images for processing. Please ensure that any images you upload follow the below guidelines specific to this IQbot.

High-Resolution:  The quality of the output data you receive is largely dependent on the input image resolution. Please provide high-resolution images to ensure that the IQbot can most effectively differentiate between features.
In-Focus:  Please produce the most in-focus images possible for analysis. Images with high background contrast will result in more accurate and useful data and overlays.
Properly Saturated:  If your submitted images are significantly undersaturated or oversaturated, it is possible that the IQbot will be unable to detect structures, or may group structures together. Please ensure that the images you upload are saturated appropriately.
Clear of Artifacts:  Please take care to avoid creating bubbles, debris, or otherwise inducing artifacts into your image. Such additional features may corrupt the resulting data.
Clear of Accessary Information:  Please do not submit images retaining any annotations, legends, timestamps, or other markings. Every part of the images that you upload will be analyzed and included in the output data.
 
 
 

If you are uncertain whether or not your images meet the quality standards for which this IQbot was validated, or have additional questions relating to image quality, please view our FAQs or contact the ImageQuantify.com support team.

 

     (216) 678-9258           help@imagequantify.com

 

Validation Information


This IQbot has been successfully validated for use, and a copy of its validation certificate accompanies the results of each order it analyzes. In accordance with general ImageQuantify.com validation procedures, this IQbot has been found to provide quantitative information equivalent to that of a manual observer, but with no variability and increased breadth of information.

 
 

The first step of the validation process was to establish the ground truth against which the IQbot’s efficacy would be compared. For the purpose of the DAB Stain Analysis IQbot, ground truth was defined on a manual observer basis. Trained observers were guided through a standardized, semi-automated process to produce output data from a specially selected validation image set. This image set represented the entire range of expected output values (e.g., different configurations, labeled targets, and DAB densities). Images were presented for analysis in a randomized order and deidentified to protect against observer biases. The observer mean for each parameter measured was accepted as ground truth.

By analyzing the inter- and intraobserver variability present in the manual observation process, acceptable criteria were established so as to compare the IQbot’s output to that of a trained replacement observer. Ultimately, the IQbot’s performance proved to offer significant upgrades over manual or semi-automated analysis techniques, and validation was considered successful. Further considerations ensured that the IQbot rejected and flagged concerning image-specific outputs and performed within operation time constraints.

 

The DAB Stain Analysis IQbot segments and quantifies regions of DAB in tissue histological sections counterstained with hematoxylin. Since the brown-colored DAB stain is an oxidative byproduct of a specific protein bound to an enzyme (i.e., HRP or horseradish peroxidase), it is agnostic to the structure and location of the target protein and serves only as a reporter. Therefore, this IQbot's output parameters may represent nuclear staining (if the DAB target is localized to nuclei) or regional staining (if the DAB target is extracellular and clustered). These output parameters include total area of DAB staining (Positive Nuclei or Region Area) and counts of the distinct regions of DAB within the image (Positive Nuclei or Region Count). Additionally, with the aid of the counterstain, the area of tissue in the image (Total Tissue Area) is also analyzed to enable normalization of the DAB staining content. Lastly, in addition to the quantitative metrics, a pseudo-colored image is generated for each image analyzed by the IQbot in which the segmented DAB staining is pseudo-colored green.

DEMO IMAGE
IQBOT APPLIED IMAGE

 

SAMPLE DATA

 

ImageTotal Tissue Area
(pixels)
Positive Nuclei or Region Count Positive Nuclei or Region Area
(pixels)
Sample 1405459 1245 156518