Human Histomorphometry Procotol

Imaging and Staining

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Trichrome Stain

Trichrome staining is common with human bone biospy because it can be used to collect referent data, osteoblast data, and osteoclast data. It's often paired with an unstained section for fluorescent labels used to measure bone formation rate and a toluidine blue stained section to measure wall width.

Digital Pathology

Since human bone biopsies can be quite large, scanning is quite helpful. It allows BIOQUANT to zoom out to the entire section so that referent data can be collected in one step. Scanning can be done either with the BIOQUANT OSTEOIMAGER scanner, one of BIOQUANT's scanning upgrade to your lab’s microscope, or in an shared imaging core.

Define the Tissue Volume

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Irregular TV Tool

The Irregular ROI Cursor helps you define the trabecular compartment as a shape that is a constant distance from the cortical shell and cut edges. In this way, the anatomy can dictate the size of the sampling area.

Rectangular TV Tool

Alternatively, if a fixed sampling area is required, the TV ROI tool creates a rectangular shape of specific dimensions that can be positioned a standard distance from the growth plate.

Collect Referent Data

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Referent Data Tool

Once the Tissue Volume is defined, BIOQUANT uses the stain color to recognize the mineralized bone and osteoid within the trabecular compartment. Automated tools correct small histology defects. Manual tools are available for larger changes.

With the bone identified, the Referent Data Tool automatically collects:

  • TV, BV, BS
  • BV/TV, BS/BV
  • Tb.N, Tb.Dm, Tb.Sp

Automatic Regions of Interest

Image Navigator

After measuring the referent data, BIOQUANT can zoom in to cellular resolution to collect additional data. While moving around in the Image Navigator, BIOQUANT will automatically update the region of interest to ensure that data are only collected within the Tissue Volume boundary.

Collect Osteoid Data

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Using the stain color to identify osteoid seams, the Osteoid Tool automatically collects:

  • OV, Ob.S, OS, O.Wi
  • OV/BV, OS/BS, Ob.S/BS

Osteoid width is measured directly at regularly spaced intervals along the osteoid surface.

Collect Osteoblast Data

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Measure Cells

Since osteoblasts cluster together and they aren't specifically stained, BIOQUANT cannot detect them automatically. Instead, a brush tool is used to mark each cell. Once the osteoblasts have been identified, BIOQUANT counts and maps them. BIOQUANT will not measure the same osteblast twice even in overlapping fields of view. Data collected:

  • Ob.N
  • Ob.N/BS

Collect Osteoclast Data

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Auto Surface

Since TRAP staining isn't common in human bone biopsy, osteoclasts must be identified manually. Using a brush tool, the osteoclasts are painted in. Then BIOQUANT can automatically measure the length of the bone associated with the osteoclast as well as the number of osteoclasts. This collects:

  • Oc.N, Oc.S
  • Oc.S/BS

Toluidine Blue Staining

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Cement lines are easier to see with toluidine blue staining so it is common to us an approximately serial section collect wall width data. Since the sections are serial, the referent data from the trichrome section can be used to normalize the wall width data.

Collect Wall Width Data

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An Alternate Use of the dLS Tool

Since the dLS tool measure the distance between two surfaces, its also a good fit for collecting wall width data. Draw once surface along the cement line and the other along the cellular surface. BIOQUANT determined the distance between them at regular intervals.

Fluorescent Staining

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Tetracycline dosing allows the section to show the surfaces that were actively mineralizing at the time the doses were given. Again using an approximately serial section allows the labeled surfaces to be normalized by the referent data from the trichrome section.

Collect BFR Data

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Auto surface: Single labeled surface

Using the color of the labeled surface BIOQUANT can automatically measure the length of labeled surfaces. A minimum label intensity as well as a minimum label size can be set to help improve the consistency of data. Manual editing may be needed to differentiate between single and double labeled surfaces. 

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Double Labeled Surface Tool

Pairs of labeled surfaces are traced manually. The dLS tool then measures the interlabel width and the length of the double labeled surface. This data is combined with single labeled surface and bone surface to automatically collect:

  • sLS, dLS
  • MS, MAR, and BFR