LDF is a continuous real time measurement, which allows the user to visualise the blood flow. Applying the laser probe onto the desired area of question uses it. The frequency of the ultrasound changes as a function of the blood speed as well as the angel at which the probe is being positioned with respect to the blood vessel (1). This is very similar to saying that a fast car coming towards you has a higher pitch sound than when it's leaving.

The procedure is sensitive but not specific since it measures all kinds of flows in the area where the laser passes through. And so, to measure the blood flow of the bone, we have to invade the probe (2).

Its great advantage is the ability of being applied clinically and accurately but not in slow blood flow (1).

It is the gold standard in the bone blood flow measurements for its high sensitivity and precision. It is widely used in measuring blood flow in bone and other soft tissues (3). The method of animal preparation and micro sphere injection with the radioactive are the same as that followed with TRAC colored micro spheres, as it is the standard technique in measurement blood flow in all kind of tissues, micro spheres in blood flow studies.

It has a great advantage in measuring a slow and a little blood flow of any tissue in the body regardless the size or percentage of the blood supply.

There are disadvantages faced when using the radioactive material: It cannot be applied clinically, the standard measurement technique using radioactive micro spheres prevents their use at institutions not able to neither dispose nor afford the cost of radioactive waste and the Radioactivity safety.

The PET is not sensitive or specific and it has a low field of view (about 4-5 mm from the probe). To measure the blood flow, it has to pass through the soft tissues.

However, the three earlier mentioned methods for measuring the blood flow in bone are accompanied by undesirable side effects, such as safety, sensitivity and specificity of the values, time effect, coast effect, permission and registration to some of them, restrictions of using a procedure, invasive procedure which of course needs sterilisation.

Having mentioned that, these methods may not be adequate for the implications they offer.

These studies were carried out using large (350 g) Sprague Dawley rats. Nine animals were anaesthetised using Midazolam and Fentanyl anaesthetic, they were placed supine on the operating table (fig.3).

Figure 1

Figure 1: Injecting the rat with the micro spheres on the operating table.

Micro spheres (1.000.000) were injected into the left ventricle via a needle inserted through the chest wall. The injections of micro spheres contained E-Z TRAC (Blue),

DYE TRAK (Yellow) or Both Micro Spheres.

After injection, the animals were allowed to stabilize for 15 min and were then sacrificed in accordance with Home Office Regulations.

All soft tissues were separated from the bones. Each limb was divided into two samples; the bone sample, which contained only the shafts of the long bones, and the muscle sample contained all the soft tissue that was removed from the long bones.

Prior to separation of the hard and soft tissues, the complete limbs were stored at -80◦C in air evacuated polythene bags (fig.5). Once the tissues had been separated from the complete limbs, after thawing, they were placed in uniquely labelled sample containers and stored at –4 degrees until processed.

Figure 2

Figure 2: Limbs in reserved in air evacuated bags

The two micro spheres species (E-Z TRAC and DYE TRAK) employ essentially the same micro spheres harvesting protocol. However, this protocol is designed for soft tissue, for bone an additional step required.

Each sample, muscle or bone, was placed in a 15 ml polypropylene centrifuge tube and pushed to the bottom, 4 ml of diluted Tissue Digest Reagent I is added to each sample.

Tubes were placed in oven at 70˚-80˚ C and allow tissue hydrolysis overnight. The following day, vortex mixing was done for 15-20 seconds. The tissue samples in each tube have been completely homogenized into suspension, with only small particles of debris visible. Diluted Tissue Digest Reagent II was added to the sample suspension to bring the total liquid volume to 14-15 ml. Tubes were centrifuged for 30 min and the supernatant was aspirated to a level slightly above each pellet.

Each sediment was re-suspended in 10 ml of diluted Micro spheres Counting Reagent by vortex mixing.

An additional aggressive digestive step for harvesting microspheres from bone, involving the immersion of the bone samples in concentrated HCl for tissue digestion.

The two micro spheres species have two different recommended counting procedures,

Manual counting using microscope, and Spectrophotometer counting after dye elution.

During the course of these studies, it became apparent that dye elution counting was the most convenient method of assessing the micro spheres population. Therefore, in addition to recommended manual counting technique we derived a dye elution technique.

The use of both micro sphere types in bone blood flow studies requires an additional digestive step to those outlined in the manufactures micro spheres harvesting protocol. This additional step requires extremely aggressive and corrosive chemicals, in particular concentrated HCl. There are two main concerns associated with the use of concentrated HCl in conjunction with micro spheres:

That concentrated HCl may alter the micro spheres structure in such a way that they are unidentifiable under the microscope.

That the interaction between HCl and the micro spheres may elute dye or product subsequent elution.

A number of simple IN-VITRO experiments were designed to answer these concerns.

Effect of concentrated HCl on micro sphere morphology was tested by adding 0.5 million micro spheres, of both species to 5 ml concentrated HCl and the mixture allowed to sit for 10 min. The acid was removed using a glass pipette and the vessel filled with 10 ml distilled water.

The tube containing micro spheres and H2O was centrifuged at 2500 rpm for 10 min and the water removed down to the 0.5 ml mark. The mixture of H₂O and micro spheres was vortex mixed and an appropriate amount of the mixture was placed on a Hemacytometer and the structure of the micro spheres observed and recorded.

Effect of concentrated HCl on dye elution was tested by adding 0.5 million micro spheres of both species to 5 ml concentrated HCl and the mixture allowed to stand. The mixture was transferred by pipette into a spectrophotometer cuvette and placed in the reading bay or spectrophotometer. The absorbance of light at the appropriate wavelength for the two micro spheres was measured and compared to a control cuvette containing only concentrated HCl, this was repeated at 10, 30, 60 min and at 72 hours.

After 72 hours, the acid was removed after centrifugation and the micro spheres exposed to DiMethyle Formamide (DMF) and the elution of dye observed by colour change and by Spectrophotometric measurement.

The in-vitro studies were carried out to determine whether the additional chemically aggressive step of adding concentrated HCl to the bone samples to facilitate digestion, has any effect on either the morphology or dye eluting qualities of the micro spheres.

On close microscopic examination (x 400), there were no morphological changes to the micro spheres. The micro spheres appeared to be intact, with no apparent micro sphere litter present. It was possible to discern the colour of the micro spheres in all cases.

Upon adding DMF to the previously acid exposed micro spheres, there was a clear leaching of yellow dye from the micro spheres into the solution. Blue dye was not apparent upon visual inspection. Analysis using the spectrophotometer confirmed that the yellow dye has leached, and that the process has no effect on the blue E-Z TRAC micro spheres

Long term exposure to DMF demonstrates that it is possible to elute dye over a long period of time from the supposedly dye-stable E-Z TRAC micro spheres. This may be useful in applications where chemical processes affect the elution in conventional DYE TRAK micro spheres, but where manual counting is confounded by low micro sphere numbers. It was clear from these studies that both micro sphere species were impervious to exposure to strong acid, insofar as in neither case was there any significant dye present at either wavelength in the acid after exposure. Furthermore, the DYE TRAK micro spheres appeared to give up most of the dye present in a short time (around 15 minutes) in the presence of DMF, with no significant further elution beyond this time.

The E-Z TRAC micro spheres did not appear to release dye over the short time scale, but prolonged exposure to DMF did result in the release of significant amounts of dye. This suggests that the E-Z TRAC micro sphere can be employed as a dye-eluting device if exposed for a long period of time. However, with no real calibration data from the manufacturer, it is difficult to quantify the number of micro spheres present as a function of the dye concentration at this stage for E-Z TRAC, but this action may be useful as a method for verifying manual counting of this micro sphere species.

Manual counts were carried out on both soft tissue and bone samples. The counts are shown in table 1 for bone and table 2 for muscle. These values are expressed as the number of micro spheres per gram of tissue in each sample.

Figure 3

Table 1: The manual counting of micro spheres in bone, group 1 was the yellow injected rats, group 2 was the blue injected rats and group 3 was the both micro spheres injected rats (NO= None Observed).

Figure 4

Table 2: The manual counting of micro spheres in muscle, group 1 was the yellow injected rats, group 2 was the blue injected rats and group 3 was the both micro spheres injected rats (NO= None Observed).

These data show quite clearly that it is possible to identify both micro spheres types in both bone and muscle, by simple microscopy. However, the numbers of micro spheres seen were fairly low in both bone and muscle and the difference between the two tissues types in terms of the number of micro spheres distributed to each tissue does not appear to confirm the expressed blood flow distribution patterns . This may be due a combination of counting errors and poor injection technique.

In all samples, there was a difference in adsorption after exposure to DMF in the yellow dye wavelength (448 nm), but no change in the blue (672 nm), when compared to control DMF, tables 3 and 4.

Figure 5

Table 3: Adsorption (ADS) of yellow (Y) and blue (B) dye in bone.

Figure 6

Table 4: Adsorption (ADS) of yellow (Y) and blue (B) dye in muscle.

It is possible to compute the number of micro spheres in the sample volume by using manufactures control sample adsorption data. In the case of yellow DYE TRAK micro spheres, 1000 micro spheres in 100 micro l DMF typically are associated with an adsorption at 448 nm of 0.068, which increases in proportion to the number of micro spheres present. Using these data, and the known weight of tissue in each sample, it is possible to express these adsorption values as number of micro spheres per gram of tissue.

For the blue E-Z TRAC micro spheres, the adsorption is 0.045 for 1000 micro spheres in 100 micro-litres of DMF. This estimation is based upon values employed for evaluation blue DYE TRAK micro sphere populations, and cannot be deemed accurate at this stage. However, the wavelength of 672, which is recommended for DYE TRAK blue, is precisely where one observes light adsorption with the blue dye associated with the E-Z TRAC species.