Lecture # 15: Bone Marrow Examination

An examination of the bone marrow is employed in the diagnosis of hematologic disorders or other diseases and other conditions not primarily affecting the blood. It is used in:

1. The diagnosis of red cell disorder

2. The diagnosis of white cell disorder

3. The diagnosis of platelet disorder

4. The diagnosis of bacterial infections, parasitic infections and storage diseases.

5. The study of response to therapy

Contraindication:       Hemophilia

Collection of specimen:

1.     Bone marrow aspiration technique with Salah, Klima needle

2.     Trephine biopsy with Jamashidi biopsy needle

3.     Surgical biopsy

 

Sites of puncture

Adult:              Sternum, iliac crests and spinous process

Infant:             anteromedial surface of the tibia

Children:         Iliac crest, spinous process

Preparation of the aspirate for examination

Marrow films:

Films can be made in a similar manner as for ordinary blood counts. Gray particles, of marrow are visible with the naked eye. They are best material for the preparation of good films and serve as landmarks for the microscopic examination of the stained smear.

a. Direct films

A drop of marrow is placed on a slide, a short distance away from one end. A film 3 to 5 cm long is made with a spreader, not wider than 2 cm dragging the particles behind but not squashing them. A trail of cells is left behind each particle.

b. Imprints

Marrow particles can also be used in the preparation of imprints. One or more visible particles are picked up with a capillary pipette, the broken end of a wooden applicator or a toothpick and transferred immediately to a slide and made to stick to it by a gentle smearing motion. The slide is air dried and stained.

c. Crush preparation

Marrow particles in a small drop of aspirate may be placed on a slide near one end. Another slide is carefully placed over the first. Slight pressure is exerted to crush the particles, and the slides are separated by pulling them apart in a direction parallel to their surface.

As the aspirated material is being spread, the appearance of fat as irregular holes in the films gives assurance that marrow and not just blood has been obtained.

Gross quantitative study

The aspirate is added to EDTA, mixed and transferred to a Wintrobe hematocrit tube with a capillary pipette. The tube is centrifuged at 2,500 rpm for 10 minutes. Four layers can be distinguished in the centrifuge: fat, plasma, myelo–erythroid (M:E) portion and erythrocytes. Their height is recorded in percentages by reading on the scale of the tube. Normally, the fat layer is 1 to 3% of the total volume and the M:E layer is 5 to 8%. The volumes of the plasma and erythrocytes layers vary considerably depending upon the degree of dilution with sinusoidal blood.

            High M:E and low fat values, in the absence of a significant leukocytosis, suggest

                        marrow hyperplasia

            Low M:E and high fat values suggest marrow hypoplasia

Histologic section

The needle biopsy and the clotted marrow particles are fixed in Zenker’s solution for 6 to 18 hours. The tissue is processed routinely for embedding in paraffin or plastic materials. Sections provide the best estimate of cellularity and a picture of marrow architecture but are somewhat inferior for the study of cytologic details.

Staining of marrow preparations

1.     Romanowsky stain

2.     Perl’s Prussion Blue Reaction for Iron

3.     Hematoxylin and Eosin for histologic sections

Examination of marrow

1. Peripheral blood examination – the one who examines the bone marrow should also carefully examine the peripheral blood on the day of the marrow study and incorporate the observations in the marrow report. This includes CBC, platelet count and reticulocyte count.

2. Cellularity of the marrow – the marrow cellularity is expressed as the ratio of the volume of hematopoietic cells of the total volume of the marrow space (cells plus fat and other stomal elements).

3. Distribution of cells

The distribution of the various cell types should be ascertained by scanning several slides under low and high, OIO power objectives. A differential count at 300 to 1000 cells should be done and then calculate the percentage of each type of cell.

4. Maturation

While examining the cells during the differential count, one should evaluate whether maturation is normal, that is, whether nuclear and cytoplasmic development is balance.

5. Presence of rare cell types or abnormal cells

In scanning the marrow, one looks for the presence of rare or unexpected cell types, like tissue mast cells, osteoblasts, osteoclasts, and metastatic neoplastic cells.

THE MYELOGRAM – DIFFERENTIAL COUNT ON ASPIRATED BONE MARROW

            Proerythroblasts                                0.5 – 1.5%

            Basophilic normoblasts                     1.5 – 3.5%

            Polychromatic normoblasts              5.0 – 10%

            Oxyphilic normoblasts                       10 – 15%

            Myeloblasts                                        0.5 – 1.5%

            Promyelocyte                                     4 – 8%

            Metamyelocyte                                  9 – 15%

            Stabs                                                   15 – 30%

            Segmenters                                         4 – 8%

            Eosinophils                                         0 – 1%

            Monocytes                                          0 – 1%

            Lymphocytes                                      8 – 20%

            Plasma cells                                        1.5 – 3.5%

            Lymphoid reticulum cells                  1 – 4%

            Macrophages                                      0 – 1%

            Sideroblasts                                        20 – 60%

            Myeloid:Erythroid ratio                     2:1 – 4:1

M:E ratio expresses the ratio of the total myeloid cells to the nucleated erythroid cells.