Histology Analysis Measurements (HFCB/HFTB/HFVB)
MicroCT Cortical MicroCT Trabecular HFCB-Histo Femur Cortical Bone HFTB-Histo Femur Trabecular Bone HFVB-Histo Vertebra Trabecular Bone
Code	Name	Short Description	Long Description
HFCB01	H/W	Height to width ratio of the distal femur (Ratio), H/W	A crude assessment of the shape of the femoral bone. It is calculated as the ratio of the fixed size ROI rectangle. The greater the ratio, the narrower the metaphyseal bone and the further the ROI extends into the diaphyseal bone.
HFCB02	Oc.N	Osteocyte density (# per µ2 ), Oc.N	The number of DAPI positive cells restricted to the cortical bone. This measurement is obtained from staining step 3 (AP activity) that utilizes DAPI as a nuclear stain. The value cannot be made with conventional µCT instruments.
HFCB03	Ct.Wth	Cortex width (µM), Ct.Wth	The average thickness of the cortical bone adjacent to the ROI expressed in µ. It is not a reliable measurement due to plane of section effect.
HFTB01 HFVB01	BV/TV	Bone volume per total volume (%), BV/TV	The percentage of area within the ROI (TV) that emits the signal indicating accumulated mineral (BV). It is a planar measurement in histology (the mean of 3 different levels) in contrast to the volumetric value of the entire ROI in µCT. Similar to µCT, the males have a greater BV than females in the femur but the difference between the two genders is much less in the vertebra. Consistent with the µCT data, there is greater variation in BV/TV measurements in male versus the female group.
HFTB02 HFVB02	Tb.N	Trabecular number (#/mm), Tb.N	The measurement is the average number of trabeculae encounter by any arbitrary line drawn within ROI. It is a calculation of the (BV/TV)/Tb.Th. The number is independent of trabecular size.
HFTB03 HFVB03	Tb.Th	Trabecular thickness (µm), Tb.Th	It is the average thickness (not length) of each bony element within the ROI. The computer recognizes the trabecular surface by the presence of the surface labeling signals and measures the thickness directly.
HFTB04 HFVB04	Tb.Sp	Trabecular spacing (µm), Tb.Sp	It is the average space length of any arbitrary line drawn within ROI. It is a calculation of the (1/Tb.N. Tb.Th.).
HFTB07 HFVB07	MS/BS	Mineralizing surfaces per bone surface(%), MS/BS	This is calculated as the surfaces containing a double label plus ½ of the surfaces containing either a single red or green label ((dLS + sLS/2)/BS)). The calculation is the traditional measure of bone surfaces under active mineral apposition. Usually females have a higher MS/BS ratio although there is significant variation of this measure within animal groups.
HFTB08 HFVB08	MAR	Mineral apposition rate(µm/day), MAR	This measurement is determined by the software as the perpendicular distance between the midpoint of each mineralization line rather than the leading edge as is performed manually. By measuring the midpoint, differences in line width usually caused by variation in section angle can be minimized. The distance in microns is divided by the day interval between the two dye injections. Usually males animals have a higher MAR but significant intragroup variation in this measurement also is observed.
HFTB09 HFVB09	BFR	Bone formation rate (µm3/µm2/day), BFR	This a calculated value obtained by MAR x MS/BS. It was intended to compare overall bone formation between two groups but it may obscure the underlying basis for differences in bone forming activity. Also the variation of this measurement is particularly high because of the high variation in the component measurements. Females usually have a higher BFR than males.
HFTB10 HFVB10	AP/BS	AP+ surfaces per bone surface (%), AP/BS	This measurement identifies all the trabecular surfaces that are AP positive which can include active osteoblasts and bone lining cells capable of resuming an active osteoblast status.
HFTB11 HFVB11	AP_L/BS	AP+ and mineral labeling surfaces per bone surface (%), AP_L/BS	Surfaces that are both AP+ and labeled (red, green, single or double) are assumed to be active bone forming surfaces, while those which are AP_NL/BS represent inactive (bone lining) surfaces. An example of a bone surface exhibiting AP and a red mineralization line is shown in figure x. Female mice usually have a higher AP_L/BS value than males.
HFTB12 HFVB12	AP_NL/BS	AP+ and non-labeling surfaces per bone surface (%), AP_NL/BS	The AP_NL/BS represent inactive (bone lining) surfaces.
HFTB13 HFVB13	AP_L/AP	Proportion of total AP that is over a labeling surface (%), AP_L/AP	When the total AP activity is low (AP_BS), the AP_L/BS measure does not accurately represent the proportion of active osteoblasts relative to the total AP signal. The converse, AP_NL/AP, is the). Females mice usually have a higher AP_L/AP than males.
HFTB14 HFVB14	AP_NL/AP	Proportion of total AP that is over a non-labeling surface (%), AP_NL/AP	AP_NL/AP is the converse of AP_L/AP.
HFTB15 HFVB15	TRAP/BS	Fraction of bone surface with TRAP label (%), TRAP/BS	This scores for TRAP activity irrespective of the cell size or the number of nuclei per cell.
HFTB16 HFVB16	TRAP_L/BS	Proportion of mineralizing surface that is covered with the TRAP label (%), TRAP_L/BS	The fraction of the labeling bone surface associated with a TRAP positive signal. This value is usually higher in females.
HFTB17 HFVB17	TRAP_NL/BS	Proportion of non-mineralizing surface that is covered with the TRAP label (%), TRAP_NL/BS	The fraction of the non-labeling (metabolically inactive) bone surface associated with a TRAP positive signal.
HFTB18 HFVB18	AP_TRAP_R_RG/BS	Proportion of bone surface where the AP, TRAP and mineralization signals are co-localized (%), AP_TRAP_R_RG/BS	We assume that the close proximity of osteoclastic and osteoblastic activity identifies a region of high bone remodeling.
HFTB19 HFVB19	TRAP_ON/TRAP	Proportion of total TRAP within the bone (marrow included) that is on the bone surface. (%), TRAP_ON/TRAP	Percentage of Total TRAP (surface and bone marrow) that is on the bone surface
HFTB20 HFVB20	TRAP_L/TRAP_ON	Proportion of TRAP on the bone surface that is depositing a mineralizing surface (%), TRAP_L/TRAP_ON	The fraction of the bone surface associated TRAP positive signal that overlies a mineralizing surface. This value is usually higher in females.
HFTB21 HFVB21	TRAP_NL/TRAP_ON	Proportion of TRAP on the bone surface that is on a non-mineralizing surface	The fraction of the bone surface associated TRAP positive signal that overlies a non-mineralizing (metabolically inactive) surface.

Histology Analysis Measurements (HFCB/HFTB/HFVB)

MicroCT Cortical MicroCT Trabecular
HFCB-Histo Femur Cortical Bone HFTB-Histo Femur Trabecular Bone HFVB-Histo Vertebra Trabecular Bone

Code

Name

Short Description

Long Description

HFCB01

H/W

Height to width ratio of the distal femur (Ratio), H/W

A crude assessment of the shape of the femoral bone. It is calculated as the ratio of the fixed size ROI rectangle. The greater the ratio, the narrower the metaphyseal bone and the further the ROI extends into the diaphyseal bone.

HFCB02

Oc.N

Osteocyte density (# per µ2 ), Oc.N

The number of DAPI positive cells restricted to the cortical bone. This measurement is obtained from staining step 3 (AP activity) that utilizes DAPI as a nuclear stain. The value cannot be made with conventional µCT instruments.

HFCB03

Ct.Wth

Cortex width (µM), Ct.Wth

The average thickness of the cortical bone adjacent to the ROI expressed in µ. It is not a reliable measurement due to plane of section effect.

HFTB01

HFVB01

BV/TV

Bone volume per total volume (%), BV/TV

The percentage of area within the ROI (TV) that emits the signal indicating accumulated mineral (BV). It is a planar measurement in histology (the mean of 3 different levels) in contrast to the volumetric value of the entire ROI in µCT. Similar to µCT, the males have a greater BV than females in the femur but the difference between the two genders is much less in the vertebra. Consistent with the µCT data, there is greater variation in BV/TV measurements in male versus the female group.

HFTB02

HFVB02

Tb.N

Trabecular number (#/mm), Tb.N

The measurement is the average number of trabeculae encounter by any arbitrary line drawn within ROI. It is a calculation of the (BV/TV)/Tb.Th. The number is independent of trabecular size.

HFTB03

HFVB03

Tb.Th

Trabecular thickness (µm), Tb.Th

It is the average thickness (not length) of each bony element within the ROI. The computer recognizes the trabecular surface by the presence of the surface labeling signals and measures the thickness directly.

HFTB04

HFVB04

Tb.Sp

Trabecular spacing (µm), Tb.Sp

It is the average space length of any arbitrary line drawn within ROI. It is a calculation of the (1/Tb.N. Tb.Th.).

HFTB07

HFVB07

MS/BS

Mineralizing surfaces per bone surface(%), MS/BS

This is calculated as the surfaces containing a double label plus ½ of the surfaces containing either a single red or green label ((dLS + sLS/2)/BS)). The calculation is the traditional measure of bone surfaces under active mineral apposition. Usually females have a higher MS/BS ratio although there is significant variation of this measure within animal groups.

HFTB08

HFVB08

MAR

Mineral apposition rate(µm/day), MAR

This measurement is determined by the software as the perpendicular distance between the midpoint of each mineralization line rather than the leading edge as is performed manually. By measuring the midpoint, differences in line width usually caused by variation in section angle can be minimized. The distance in microns is divided by the day interval between the two dye injections. Usually males animals have a higher MAR but significant intragroup variation in this measurement also is observed.

HFTB09

HFVB09

BFR

Bone formation rate (µm3/µm2/day), BFR

This a calculated value obtained by MAR x MS/BS. It was intended to compare overall bone formation between two groups but it may obscure the underlying basis for differences in bone forming activity. Also the variation of this measurement is particularly high because of the high variation in the component measurements. Females usually have a higher BFR than males.

HFTB10

HFVB10

AP/BS

AP+ surfaces per bone surface (%), AP/BS

This measurement identifies all the trabecular surfaces that are AP positive which can include active osteoblasts and bone lining cells capable of resuming an active osteoblast status.

HFTB11

HFVB11

AP_L/BS

AP+ and mineral labeling surfaces per bone surface (%), AP_L/BS

Surfaces that are both AP+ and labeled (red, green, single or double) are assumed to be active bone forming surfaces, while those which are AP_NL/BS represent inactive (bone lining) surfaces. An example of a bone surface exhibiting AP and a red mineralization line is shown in figure x. Female mice usually have a higher AP_L/BS value than males.

HFTB12

HFVB12

AP_NL/BS

AP+ and non-labeling surfaces per bone surface (%), AP_NL/BS

The AP_NL/BS represent inactive (bone lining) surfaces.

HFTB13

HFVB13

AP_L/AP

Proportion of total AP that is over a labeling surface (%), AP_L/AP

When the total AP activity is low (AP_BS), the AP_L/BS measure does not accurately represent the proportion of active osteoblasts relative to the total AP signal. The converse, AP_NL/AP, is the). Females mice usually have a higher AP_L/AP than males.

HFTB14

HFVB14

AP_NL/AP

Proportion of total AP that is over a non-labeling surface (%), AP_NL/AP

AP_NL/AP is the converse of AP_L/AP.

HFTB15

HFVB15

TRAP/BS

Fraction of bone surface with TRAP label (%), TRAP/BS

This scores for TRAP activity irrespective of the cell size or the number of nuclei per cell.

HFTB16

HFVB16

TRAP_L/BS

Proportion of mineralizing surface that is covered with the TRAP label (%), TRAP_L/BS

The fraction of the labeling bone surface associated with a TRAP positive signal. This value is usually higher in females.

HFTB17

HFVB17

TRAP_NL/BS

Proportion of non-mineralizing surface that is covered with the TRAP label (%), TRAP_NL/BS

The fraction of the non-labeling (metabolically inactive) bone surface associated with a TRAP positive signal.

HFTB18

HFVB18

AP_TRAP_R_RG/BS

Proportion of bone surface where the AP, TRAP and mineralization signals are co-localized (%), AP_TRAP_R_RG/BS

We assume that the close proximity of osteoclastic and osteoblastic activity identifies a region of high bone remodeling.

HFTB19

HFVB19

TRAP_ON/TRAP

Proportion of total TRAP within the bone (marrow included) that is on the bone surface. (%), TRAP_ON/TRAP

Percentage of Total TRAP (surface and bone marrow) that is on the bone surface

HFTB20

HFVB20

TRAP_L/TRAP_ON

Proportion of TRAP on the bone surface that is depositing a mineralizing surface (%), TRAP_L/TRAP_ON

The fraction of the bone surface associated TRAP positive signal that overlies a mineralizing surface. This value is usually higher in females.

HFTB21

HFVB21

TRAP_NL/TRAP_ON

Proportion of TRAP on the bone surface that is on a non-mineralizing surface

The fraction of the bone surface associated TRAP positive signal that overlies a non-mineralizing (metabolically inactive) surface.