Bone Marrow Examination

 

UNIT 4 BONE MARROW EXAMINATION

BONE MARROW:

Bone marrow is soft, spongy, gelatinous tissue that fills the centers of bones known as medullary cavities. Bone marrow is highly vascular meaning that it is enriched with blood vessels and capillaries. Each day bone marrow produces hundreds of billions of new blood cells.Inadult humans, bone marrow is primarily located in the ribs, vertebrae, sternum, and bones of the pelvis. Bone marrow comprises approximately 5% of total body mass in healthy adult humans, such that a man weighing 73 kg will have around 3.65 kg of bone marrow.

Types of Bone Marrow

1. Red Bone Marrow

It is also known as myeloid tissue. Its main function is to produce blood cells. In addition, it also helps to remove old cells from blood circulation (spleen and liver also remove old and damaged blood cells from the blood).From birth to early adolescence, the majority of our bone marrow is red marrow. With increasing age, red marrow is replaced by yellow marrow. In adults, red marrow is confined mostly to bones of the skull, pelvis, spine, ribs, sternum, vertebrae, shoulder blades, long bones of the arms (humerus) and legs (femur and tibia).

    All other spongy bones and central cavities of the long bones are filled with yellow marrow. Red bone marrow consists of a delicate, highly vascular fibrous tissue that contains hematopoietic stem cells. These are blood-forming stem cells and are of two types: myeloid stem cells and lymphoid stem cells. These cells develop into red blood cells, white blood cells, and platelets.

2. Yellow Bone Marrow

It is also known as fatty bone marrow as it consists mainly of fat cells. It is found in spongy bones and in the shaft of long bones. Its main function is to store fat. It also helps to maintain the correct environment for the bone to function. It is composed of hematopoietic tissue that has become inactive. In conditions such as massive blood loss, when blood cells become low, yellow marrow can be converted to red marrow in order to produce more blood cells.

Bone Marrow Stem Cells

The bone marrow contains two types of stem cells:

1. Mesenchymal Stem Cells

These are also known as marrow stromal cells. They are found in yellow marrow. These produce non-blood cell components of marrow including

a. Fat cells (adipose tissue)

b. Chondrocytes (cartilage)

c. Fibroblasts (fibrous connective tissue)

d. Stromal cells (that support blood formation)

e. Osteoblasts (for bone synthesis)

f. Osteoclasts (for bone restoration)

2. Hematopoietic Stem Cells

They are found in the red marrow and are responsible for the production of blood cells. They are immature cells that can turn into a number of different types of blood cells.

The different types of hematopoietic stem cells have different regenerative capacity and potency. They rapidly multiply and undergo maturation through different stages to produce mature blood cells. Millions of blood cells are produced each day.

The process of development of different blood cells from the stem cells is known as hematopoiesis.

Functions of Bone Marrow

1. Hematopoiesis

Bone marrow is the site for hematopoiesis. All the blood cells are produced in the bone marrow. However, some white blood cells after being produced in the bone marrow migrate to other lymphoid organs such as the spleen, lymph nodes, and thymus gland for maturation.

2. Stem Cells

Bone marrow acts as a reservoir of stem cells – both hematopoietic and mesenchymal.Hematopoietic stem cells are harvested from bone marrow or peripheral blood for the purpose of bone marrow/ stem cell transplantation.

3. Bone Marrow Barrier

The hematopoietic stem cells are able to cross the bone marrow barrier and hence can be harvested from blood.

In a few diseased conditions, the immature cells may get released into the circulation. These include conditions like hemolytic anemia in which bone marrow produces an increased number of cells to compensate for the hemolysis or in leukemias when a large number of immature cells are being produced and there is a failure of maturation.

4. Lymphatic Role

The red bone marrow is an important element of the lymphatic system since it produces lymphocytes. Both the bone marrow and thymus constitute primary lymphoid tissue as they are involved in the production and early selection of lymphocytes.

 

 

 

 

 

 

 

 

 

 

BONE MARROW ASPIRATION IN CHILDREN AND ADULTS:

Bone marrow examination refers to the pathologic analysis of samples of bone marrow obtained by bone marrow biopsy (often called trephine biopsy) and bone marrow aspiration.  It is also known as lumbar puncture technique. Bone marrow examination is used in the diagnosis of a number of conditions, including leukemia, multiple myeloma, lymphoma, anemia, and pancytopenia. The bone marrow produces the cellular elements of the blood, including platelets, red blood cells and white blood cells.

Bone marrow samples can be obtained by aspiration and trephine biopsy. Sometimes, a bone marrow examination will include both an aspirate and a biopsy. The aspirate yields semi-liquid bone marrow, which can be examined by a pathologist under a light microscope and analyzed by flow cytometry, chromosome analysis.

Site of procedure

Bone marrow aspiration is usually performed on the back of the hipbone, or posterior iliac crest. An aspirate can also be obtained from the sternum (breastbone). For the sternal aspirate, the patient lies on their back, with a pillow under the shoulder to raise the chest.

    While spinous process aspiration is frequently done in a lumbar puncture position and on the L3-L4 vertebrae.

The preferred site for obtaining bone marrow in childrenis the posterior superior iliac crest because it contains the most cellular marrow; there are no vital organs in close proximity.

When the procedure is performed by an inexperienced technician; there is also a risk of fracturing the bone.  Bone marrow aspiration (BMA) and bone marrow trephine biopsy (BMTB) must be performed only by experienced health care providers who have been well-trained in the technique

Anesthesia is used to reduce surface pain at the spot where the needle is inserted. Pain may result from the procedure's insert to the marrow, which cannot be anesthetized.

Materials used for aspiration:

a) 2% lignocaine (2-5ml) (anathesia)

b) Syringes 2ml, 5ml

c) Needles 21, 22, 25 gauge

2 types of needles are used:

Jamshidi needle, klima needle

d) EDTA

e) Clean glass slides.

Procedure:

1. The patient is asked to lie on their abdomen or on their side

2. The skin is cleansed, and a local anesthetic is  injected to numb the area.

3. Patients may also be pretreated with analgesics and/or anti-anxiety medications, although this is not a routine practice.

4.An aspirate needle is inserted through the skin using manual pressure and force until it abuts the bone.

5. Then, with a twisting motion of clinician's hand and wrist, the needle is advanced through the bony cortex (the hard outer layer of the bone) and into the marrow cavity.

6. Once the needle is in the marrow cavity, a syringe is attached and used to aspirate ("suck out") liquid bone marrow.

7. A twisting motion is performed during the aspiration to avoid excess content of blood in the sample, which might be the case if an excessively large sample from one single point is taken. Subsequently, the biopsy is performed if indicated.

8. A different, larger trephine needle is inserted and anchored in the bony cortex. The needle is then advanced with a twisting motion and rotated to obtain a solid piece of bone marrow. This piece is then removed along with the needle.

9. The entire procedure, once preparation is complete, typically takes 10–15 minutes.

10. After the procedure is complete, transport the sample in container and immediately transfer it to the lab or if inside lab, prepare 10-15 smears of aspirated bone marrow. Work should be done quickly to avoid marrow clotting.

11. Transfer the left over marrow in EDTA tube and mix.

12. The patient is typically asked to lie flat for 5–10 minutes to provide pressure over the procedure site. After that, assuming no bleeding is observed; the patient can get up and go about their normal activities.

13. Paracetamol or other simple analgesics can be used to ease soreness, which is common for 2–3 days after the procedure.

14. Any worsening pain, redness, fever, bleeding or swelling may suggest a complication. Patients are also advised to avoid washing the procedure site for at least 24 hours after the procedure is completed.

Staining: Stain bone marrow films or smear with any of Romanowsky stain (Leishman, Giemsa stain)

 

Preparation of  the bone marrow smear.

 (A) When the bone marrow aspiration needle is in place, use a 20 ml syringe to draw 1 ml of aspirate.

(B) Place a few drops of aspirate on a slide held vertically to facilitate visualisation of the bone marrow spicules.

(C) Using the edge of a second slide, separate and isolate a few bone marrow spicules from the rest of the sample.

(D) Transfer these to a third slide.

(E) Without applying pressure, distribute the spicules in the centre of the slide.

(F) Place the second slide across the third slide to form a cross with the spicules in the centre.

(G) Gently but quickly slide the second slide over the third slide to extend the spicules along its length.

(H) Make sure the aspirate is spread evenly over the entire slide.

 (I) Allow to dry for 5 min, and then send it for staining.

Staining of bone marrow smear by using Romanowsky stains:

I. Wright stain

Wright's stain is a hematologic stain that facilitates the differentiation of blood cell types. It is classically a mixture of eosin (red) and methylene blue dyes. It distinguishes easily between blood cells; it became widely used for performing differential white blood cell counts, which are routinely done when conditions such as infection or leukemia are suspected.

Procedure:

1. Place 1 ml of the Wright Stain Solution upon the smear 1 – 3 minutes.

2. Add 2 ml distilled water or Phosphate buffer pH 6.5 and let stand twice as long as in step 1.

3. Rinse stained smear with water or the Phosphate buffer pH 6.5 until the edges show faintly pinkish-red.

4. For a Giemsa appearance stain 10 minutes in one volume Wright Stain and 4 volumes Phosphate buffer, pH 6.5.

5. Make it dry very carefully.

6. Observe under oil immersion microscope.

STAIN RESULTS

1. Red blood cells red to pink

2. Neutrophils dark purple nuclei, pale pink cytoplasm, reddish-lilac small granules

3. Eosinophils blue nuclei, pale pink cytoplasm, red to orange-red large granules

4. Basophils purple to dark blue nucleus, dark purple, almost black large granules

5. Lymphocytes dark purple to deep bluish purple nuclei, sky blue cytoplasm

6. Platelets violet to purple granules

II. GiemsaStain

Principle: it is composed of both the Acidic and Basic dyes, in relation to affinities of acidity and basicity for blood cells. Azure and methylene blue, a basic dye binds to the acid nucleus producing blue-purple color. Eosin is an acidic dye that is attracted to the cytoplasm and cytoplasmic granules which are alkaline-producing red coloration. The stain must be buffered with water to pH 6.8 or 7.2, to precipitate the dyes to bind simple materials. Basically, Giemsa stain is a differential stain which is made up of a combination of reagents (Azure, Methylene blue, and Eosin dye)

Procedure:

1. Add a thick smear of blood and air dry for 1 hour on a staining rack.

2. Put the thick blood smear slide into diluted Giemsa stain.

3. Wash the smear by dipping in in buffered water of distilled water for 3-5 minutes

4. Leave it to dry

5. Observe under oil immersion microscope.

Results

a) The Cytoplasm and cytoplasmic granules of blood cells appear red in color while the nucleus appears blue-purple in color.

b) The erythrocytes will appear pink in colour

c) Eosinophil’s will have a blue-purple nucleus, a pale pink cytoplasm, and orange-red granules.

d) Neutrophils will appear purple-red nucleus and a pink cytoplasm.

e) Basophils will have a purple nucleus and bluish granules.

f) Lymphocytes have a dark blue nucleus and a light blue cytoplasm.

g) Monocytes will have a purple nucleus and a pink cytoplasm.

h) Platelets will have purple granules.

DIFFERENTIAL CELL COUNT ON BONE MARROW (MYELOGRAM):

1. Many workers perform count on marrow films by presenting the data in the form of myelogram and expressing the cell type in percentage.

2. It is necessary to choose a proper area for differential count.

3. Estimate the marrow the hyper-cellular, normo-cellular and hypo-cellular.

4. Select an area where cellular area of film is highly spread and stained.

5. The cells should be examined with oil immersion objective.

6. In adults, about 10% of nucleated cells are lymphocytes.

7. Their number may be higher in children.

8. For megakaryocytes, monocytes and plasma cells both number and morphology should be observed.

 

 

 

 

 

 

Collection of Bone Marrow trephine biopsy specimens

The BM trephine biopsy may be performed either before or after the aspirate. The length of the core from an adult should be at least 2 cm. A shorter core (e.g. 1 cm) may sometimes contain sufficient diagnostic information. The biopsy specimen shrinks by approximately 20% after processing. However, the larger the amount of tissue that is biopsied, the greater is the likelihood of a focal lesion (e.g. lymphoma, metastatic tumour, granulomas) being detected. Bilateral trephine biopsies may be performed to increase the yield of detecting focal lesions.The specimen should be handled gently to avoid crush artifact and distortion. Touch imprints should be made from the trephine biopsy prior to placing in fixative.Imprints are made by gently touching the fresh unfixed core on the slide, or the slide on the core. The imprints are fixed and stained using the same method as for aspirate smear