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Clinical Significance

Some compounds can affect the number and morphology of cellular elements in blood by altering their production or maturation within the bone marrow. These effects may be detected initially in peripheral blood as a non- or poorly regenerative anemia, leukopenia, leukemia or thrombocytopathy. Some processes are associated with characteristic morphologic changes. More sensitive and specific information about the effects of a treatment on bone marrow cells can be obtained by direct evaluation of a bone marrow sample. This procedure permits determination of total and differential cellularity and examination of morphology from a reproducible site. Often, the findings are consistent with or suggestive of a particular type of effect and can be used to design studies to explore specific mechanisms of action.



Principle

To evaluate bone marrow cellularity and morphology, a suspension of cells is prepared from the femur of a rat or mouse. The bone is dissected and both the proximal and distal ends are removed at the epiphyseal plates. A buffer solution is injected gently into one end of the shaft. This procedure flushes the marrow out through the opposite end and into a collection vessel. Additional processing provides a single cell suspension and breaks up bone spicules. The suspension is used to measure total cell counts, to make cytologic preparations for differential cell counts and morphologic evaluation, and, if desired, to perform other laboratory procedures.

If properly prepared, cellular suspensions of the entire bone marrow are produced and total cell counts per femur permit the detection of treatment effects on cellularity. Cytocentrifuge preparations are representative of the population of cells from the entire femur and not from a restricted sampling site. Cytological detail is optimal. Both femora may be processed to obtain two separate samples for a more confident quantitation of the nucleated cells. Or, the other femur may be used for routine histological sectioning and staining.

Materials and Equipment

Magnetic mixer and stirring bar

Hanks' Balanced Salt Solution (HBSS) without phenol red, calcium- and magnesium-free.

Tripotassium EDTA (K3EDTA)

Bovine serum albumin (BSA)

Gentamicin sulfate

Flask or beaker 600 mL or larger

pH meter with conc HCl and NaOH

Surgical instruments scalpels, safety razor blades, scissors, rongeurs, tissue forceps

Necropsy board wax or wooden

Syringes 5 mL (rats) or 3 mL (mice), disposable plastic

Needles to fit syringes: 18-19 & 21 gauge (rats), 22-23 & 25 gauge (mice)

Test tubes 5 mL, plastic only. NOTE: If 5 mL tubes are too narrow and the needle on the syringe cannot touch the bottom of the tube, wider 10 or 15 mL plastic tubes may be used initially, and then the solution may be transferred back to the 5 mL tubes..

Test tube racks

Beaker or bucket of ice

Cell or particle counter

Automatic pipetters and tips Two 200 mL capacity, hand held

Microscope slides 3 x 1 inch, frosted at one end, good quality

Cytocentrifuge & supplies

Slide stainer & stain

Mounting medium eg, Permount (Fisher Scientific, toluene based) or Micromount (Surgipath, xylene based)

Glass cover slips 22 x 40 mm

Sauna or drying oven (optional)



Supplemented Buffer

Description

Hanks' Balanced Salt Solution (HBSS) without phenol red indicator

Supplemented with bovine serum albumin and K3EDTA

Gentamicin sulfate*



Preparation

Allow at least an hour for preparation of the supplemented buffer.

A flask or beaker of the HBSS buffer is placed on a magnetic mixer and stirred as supplements are added. For every 100 mL HBSS add:

0.15 g K3EDTA NOTE: Do not use Na2EDTA because of its poor solubility in solutions at physiological pH.

5 g bovine albumin*

0.1 mL gentamicin sulfate*

*Gentamicin sulfate is added only if the suspensions will be used for preparation of cell cultures. If the addition of albumin or gentamicin will interfere with other procedures (e.g., quantitation of cellular enzymes, cytochemistry), these may be omitted.

Add the bovine albumin in small increments. The powder does not dissolve readily.

Adjust the pH of the buffer to 7.4 using either concentrated HCl or NaOH.

Storage and Stability

While fresh buffer provides the best results, the supplemented solution may be stored at 4°C for one week.



Sample Collection and Preparation

To ensure viability of bone marrow cells, the freshly dissected femur should have both distal and proximal ends intact and be processed within 10 minutes of death of the animal. Morphological differentiation between cell stages and cell lines becomes difficult as viability of the cells decreases. Bone marrow cells should be treated gently. Aggressive flushing from the femur will disrupt cellular membranes, resulting in low counts and poor cytological detail on the stained preparations.

Preparation of syringes

If several animals will be used, syringes can be prepared an hour or so ahead of time to facilitate processing.

Attach a needle to a 5.0 mL syringe, remove the cap and draw up the supplemented HBSS into the syringe.

Rats 18 or 19 gauge needle 3.0 mL buffer

Mice 22 or 23 gauge needle 1.5 mL buffer

Replace the cap of the needle and store the prepared syringes in a beaker of ice. Have other gauge needles (21 for rats, 25 for mice) available at the dissection area.



Femur removal

NOTE: The femur should be processed within 10 minutes after death of the animal.

Euthanize the animal and dissect the femur.

Using a pair of forceps and a scalpel, scrape away as much muscle and fascia from the bone as possible.

Using a scalpel or single edge safety blade, cut off the proximal end of the femur by slicing between the lesser trochanter and the neck (figure 1). Rongeurs may be used if bone is difficult to cut.

Gently remove the distal joint capsule (not shown) with a pair of forceps, being careful not to break the distal articular cartilage.

Cut off the distal epiphysis at the level of the largest intercondyloid fossa.

Hold femur with the forceps and puncture both ends of the bone with the syringe needle to produce a small opening at each site.

Using a pair of forceps, hold femur tightly over a 5.0 mL test tube and place the tip of the needle (which is mounted to a filled syringe) into the puncture site at the proximal (hip) end of the bone. Inject the buffer pressure to the plunger. The marrow and buffer will be flushed out the distal end of the bone and into the test tube.

Cells must be treated gently at all times.

Do not vortex or shake!

Aspirate 3.0 mL of air into the syringe and gently push the air through the femur. This will remove any buffer and marrow remaining in the needle or in the femur.

Remove the needle and syringe from proximal end of femur.

Draw the 3.0 mL of buffer containing the bone marrow from the test tube back into the syringe.

Using the forceps, hold the femur (distal end up) over the same 5.0 mL test tube and place the tip of the needle into the hole in the distal end of the bone. Gently push the suspension back through the femur and into the test tube.

Again, aspirate 3.0 mL of air into the syringe and slowly push it through the femur to remove remaining buffer and marrow.

Using the same needle and syringe, aspirate the bone marrow suspension from the test tube into the syringe. Be certain that all clumps of marrow that may have settled to the bottom of the tube are aspirated.

Place the flat (beveled) side of the needle against the inner wall of a the test tube and gently expel the bone marrow suspension into the tube. This will break up clumps of marrow and produce a suspension of single cells. If clumps are still visible, repeat this step, using a smaller gauge needle. Replace the needle if it becomes blocked. Cap the tubes.

This suspension is used for quantitation of cells and preparation of cytocentrifuge preparations. Process cells right away. If some time must elapse before processing, place the capped tubes in cool or slushy ice water. Do not store the tubes directly on ice.



Quantitation of Nucleated Cells

The suspension is assayed using the WBC channel of the hematology analyzer to determine total number of nucleated cells. The count is expressed as:

# cells x 103/mL

Rats Because the total volume of suspension is 3 mL, this count is multiplied by a factor of 3000 (that is, the # of mL in 3 mL) to convert the count to the number of nucleated cells per femur.

Mice Because the total volume of suspension is 1.5 mL, this count is multiplied by a factor of 1500 (that is, the # of mL in 1.5 mL) to convert the count to the number of nucleated cells per femur.

The final total femur count will be in millions (eg, 18.0 x 106)
Cytospin 2 (Cytocentrifuge) Preparations

After cells have been counted, dilute the cell suspension to a concentration of 50,000 cells per 200 mL of solution, using HBSS as the diluent.

Label microscopic slides with appropriate information (animal and study ID numbers, date) and prepare sample chambers.

Determine that sample chambers are thoroughly dry before use.

Pipette 200 mL of diluted cellular suspension into each sample chamber. To prevent the solution from being absorbed onto the filter card before centrifugation is started, hold the chamber in an upright position.

Place cover on sample tray.

Place sample tray in the Cytospin 2 and centrifuge at 500 RPM for five minutes.

When the Cytospin 2 has stopped, remove sample tray. Carefully remove slides from sample chambers without disturbing the small circle of cells adhering to the glass. Allow the slides to air dry for 10 minutes.

Stain the dried preparations as soon as possible using an automated slide stainer and Wright-Giemsa stain pack. The slides may also be stained manually, using the Wintrobe method and a commercial Wright-Giemsa preparation.

Cover slip the slides using a synthetic mounting medium.



Important Comments

Because macrophages adhere to glass, all materials used for this procedure should be plastic (polystyrene).

If the cells are to be cultured, precautions should be taken to keep the specimen sterile throughout the procedure. The work should be done using a tissue culture hood with laminar air flow and sterile materials and instruments.



References

Luster MI, Boorman GA, Korach KS, Dieter MP, Hong L. Mechanisms of estrogen-induced myelotoxicity: evidence of thymic regulation. J. Immunopharmacology 1984; 6(No. 4):288.

Boorman GA, Hong LH, Dieter MP, Hayes HT, Pohland AE, Stack M, Luster MI. Myeloltoxicity and macrophage alteration in mice exposed to Ochratoxin A. Toxicology and Applied Pharmacology 1984; 72:305.

Foster R, Metcalf D, Robinson WA, Bradley TR. Bone marrow colony stimuulating activity in human sera. British Journal of Haematology 1968; 15:148.

Hi,
Thank you for posting the protocol.
Did you know that there is a specific place to post them.
Go into the protocol tab, and post them over there.
Thanks again.
Guy

It is unnecessary, at least in mice, to clean the femur and flush the buffer through from one end to the other. Simply, remove the leg, cut, and flush with a needle that has been bent in a V-shape while holding the leg over a tube. It is much faster.