URGENT Question regarding Percent of Area%.

valpal

I need to measure the amount of positive (DAB positive) in IHC pictures. After I assign class positive and class negative (the nuclei), I need to select background in order to count the amount of negative and positive pixels. How is the percent of area calculated? what it is Area (pixel^2)? I need to understand how the background area is influencing the percent of positive and how should I take into account the negative area for the analysis. thanks please let me know as soon as possible

YuriG

Hi Valentina,

Ok, if you just want to measure total Area of your staining, then it's reported by the Sum statistical parameter of Area measurement. The Area is measured in calibrated units, if you image has calibration, otherwise in pixels. You can use grouping by Class and activate Sum as Totals to see Sum values for both classes. 

To check relative area of staining on the image, you can use Percent Area(%) (100% is whole image), it can also be reported as Sum per class.

Yuri

YuriG

There are several Percent area measurements, you can check the description in the measurement tooltip.

1. Percent area (%) and Percent area ratio - use total ROI area.
2. Percent area parent - uses the area of parent object.

Parents can be created using Class 1 and then you can count Nuclei using Class 2 or higher. Use "Preserve existing data" option in the Count options. "Parent Name" will report the name of the parent object and "Percent area parent" will report the ratio of the object area to parent area.

Check some other links related to Parent/Child measurements: 

http://forums.mediacy.com/discussion/comment/964/#Comment_964

http://www.mediacy.com/index.aspx?page=ViewSolution&solid=1732
http://www.mediacy.com/index.aspx?page=IP_Premier_Colocalization

 
Regards,

Yuri

valpal

Hi Yuri.
this is not my question I think... I am not using parent/child measurements. I have assigned class negative and class positive and background. Then I press count and I get the data table with different parameters (percent of area for both classes for example).
Is that calculated by %positive area=positive pixels area/(background pixels+positive pixels area+negative pixels area) ?

YuriG

No, that is not how it's calculated. In my previous comment I explained the ways different "Percent area" measurements are calculated.

What do you actually want to measure?

Yuri

valpal

I need to measure the amount (area) of brown staining (my protein), for comparisons between groups. 
In order to be precise and better distinguish brown from blue (which is nuclear staining) as my protein can be both non nuclear and nuclear, I was suggested to assign 2 classes plus the background (I used the SMART tool). The assignments of the 2 classes are pretty accurate actually, but Now I don't know what parameter to use from the data table. 
valentina

valpal

Alright but this means that the relative (class 1 for ex) area of staining is = relative / total in % and Total area is= %of Class I + % of Class II + Background 
Or?

YuriG

Yes, if Background is everything outside the Class 1 and Class 2 and class 1 and 2 are not overlapped, as in your case with smart segmentation.
In general, with Threshold segmentation, classes can overlap, also you could have a separate segmentation class "Background" which is not the whole image, so Class1 + Class2 +Background may not give the total image area.

Liudas_Dabkevičius

Dear Yuri

i was following your advice:

1. Percent area (%) and Percent area ratio - use total ROI area.
2. Percent area parent - uses the area of parent object.
Parents can be created using Class 1 and then you can count Nuclei using Class 2 or higher. Use "Preserve existing data" option in the Count options. "Parent Name" will report the name of the parent object and "Percent area parent" will report the ratio of the object area to parent area.

But i do not see my object area ratio to parent area (class1) 

the ratio is reported to whole immage.

YuriG

Hello Liudas,

As I can see from the screenshot that you don't have Class 1 parents.

The Count must be done in 2 steps:
1. Select Class 1 (or reset Classes), switch off "Preserve existing data". Segment parent, click Count. - you will get your parents.
2. Activate "Preserve existing data", "Use as outlines", segment children, click Count - you will get correct parent/child associations and measurements.

Regards,

Yuri

Liudas_Dabkevičius

So it is not possible to do it with one count?

YuriG

It is also possible to do that in one Count, in that case you have to reset classes, switch off "Preserve existing data", segment parent objects as Class 1 and children as Class 2, Class 3,..., click Count.

Yuri

Liudas_Dabkevičius

well in taht case my class 1 is not detected at all i cann ot find the reason

YuriG

You don't have objects of Class 1. Try to reset classes in the Count/Size ribbon and in the Threshold tool. Then segment ONLY parents and click Count (as a test) - check that these objects have Class 1. If the objects have Class 1 then add Child classes and click Count again.

Yuri

Liudas_Dabkevičius

No somethinh is not right. I reset the classes and i can count Class1 seperately, but when i add child Classes (2,3,4) Class1 is not detected and counted..

i noticed later that it is buggy. if i add 2 child classes it counts everything well.
if i add 3 child classes it does not count..:((

YuriG

Maybe it's segmentation ranges, be sure you activate "Allow overlap" (in the Show drop-down) if children are inside the parent.

YuriG

Liudas, 

If you still can't find the problem, send me the test image and segmentation ranges (saved from the Threshold tool) and I will check them.

Yuri

Liudas_Dabkevičius

Thanks'

After restartin softweare began to work.

So if this simple solution works i do not see any benefits of using Child/Parent aplication from now.

thanks

YuriG

Yes, if your objects can be simply segmented using one tool, then you don't need the app.
The app provides a workflow to segment parent and multiple child classes using different segmentation options for each parent and child class, so it can handle more complex cases.

Yuri