Dr Piyush Tailor

Purpose of examination: Bone marrow aspiration smears are primarily used for differential counts and assessment of the myeloid: erythroid ratio. They are the most suitable preparation for studying cellular details and the maturation process of the hematopoietic cells and for characterizing the abnormal cells. Aspiration is particularly useful in investigating patient with suspected iron deficiency anemia, anemia of chronic disease, megaloblastic anemia and acute leukemia. Marrow smears are also useful in the evaluation of iron stores and are the primary source of special cytochemical stains.

Bone marrow aspiration is invasive procedure that requires complete knowledge of patient including primary diagnosis with differentials considered, clinical indications and peripheral smear findings.

Principle of examination: A bone marrow sample obtained by aspiration needle is placed on a slide; smears are prepared; are air dried, fixed in methanol and stained with Giemsa stain. Smears are examined under scanner and low power to assess cellularity, megakaryocyte, any large atypical cells; then differential count, M: E ratio, maturation abnormality should be evaluated along with storage iron.

Performance specification:

Not applicable.

Sample type required:

Smears prepared from aspirated marrow

Slides are fixed in methanol and stained with Giemsa

Preservative needed;

Not applicable

Reagents required:

Giemsa stain

Methanol (acetone free)

Calibration method:

Not applicable

Detailed work bench instruction:

Sites of aspiration:

Posterior superior iliac spine

Anterior superior iliac spine

Sternum

Tibial tuberosity (in infants)

Method:

• Informed written consent is taken of the patient by clinician and then patient is atropinised by giving 1 ml intramuscularly.
• After 20 min of atropinising, patient is given lateral supine knee chest position.
• Posterior superior iliac spine is felt and then cleaned with spirit, betadine and spirit.
• Local injection of 2ml lignocaine is given sub cutaneously/ local tissue and sub-periosteally.
• Bone marrow aspiration needle guard is adjusted and then perpendicularly inserted into the bone.
• When give away sensation is felt, pull out the trocar and checked by placing the trocar on the gloves for blood.
• When blood is seen on checking on gloves, 10cc syringe is attached to the posterior side of the bone marrow aspiration needle and slight negative pressure is given.
• As soon as 0.1ml marrow material is aspirated the syringe is detached out from needle and 2^nd syringe inserted.
• The marrow material is spread on slides (if marrow is diluted with blood reaspirate excessive blood with the help of the syringe).
• When particles are appreciated further aspiration is avoided and the needle is withdrawn out and the site is strongly pressed with the help of cotton swab for 10 minutes.
• Slides are allowed to quick drying and then labeled.
• Fixation is done with 99% methanol by placing the slides in the coplin jar for 20 min and then again dried.
• 8 slides are fixed and rest all are kept unfixed for further special staining.
• 2 slides are stained by Giemsa 1:6.5 and 1:7.5 dilution with 6.8ph buffer for 15 minutes.
• In case of anemia iron stain is done on one slide.
• Parallel staining of peripheral smear of the patient is done.
• PAS and Sudan stains are done as and when needed.

Method of iron stain:

• Mix 22.5ml of 11N HCL and 27.5ml of Dist water to make 50 ml solution of 5N HCL
• Incubate the solution for 1 hr
• Dissolve 0.5gm Potassium Ferrocynide in 25 ml of Dist water
• Mix this solution with 25 ml of 5N HCL
• Keep the slides in slide rack containing prepared solution for 30min in incubator
• Wash the slides in running tap water for 15 min
• Dip the slides in safaranin solution for counterstain
• Wash with dist water
• Dry the slides

• A bone marrow film should first be examined macroscopically to make sure that particle or fragments are present.

Microscopic examination of bone marrow

• On Low power (x10) examine for the following
  • Determine cellularity
  • Identify megakaryocytes
  • Look for clumps of abnormal cells
  • Identify macrophages
• On High & oil field (x40, x100) examine for the following
  • Identify all stages of maturation of myeloid and erythroid cells.
  • Determine the M:E ratio
  • Perform a differential count
  • Look for areas of BM necrosis.
  • Assess the iron content.
• Cellularity is assessed from marrow particles by visual evaluation of fat and cells keeping in mind that in infants marrow is 95-100% cellular, in children it is 75-80% cellular, while in adults cellularity decreases to 50-60%. In old age 20-40% cellularity is taken as normal
• The M:E ratio is the ratio of all granulocytic plus monocytic cells (Myeloid) to all erythroblasts (Erythroid).
• For all bone marrow aspirates examined, the report should specify the M:E ratio and the percentage of lymphocytes and plasma cells.
• A differential count of at least 200-300 cells should be performed.
• If there is any borderline abnormality, e.g. in the number of blasts, lymphocytes or plasma cells, a 500 cell differential count should be performed.
• Only after the bone marrow has been carefully assessed on low and medium power should the X100 oil be used to assess cellular detail.
• Proerythroblast: This is a large cell with a round nucleus and a finely stippled chromatin pattern. Nucleoli are sometimes apparent.The cytoplasm is moderately to strongly basophilic.There may be a paler staining area of cytoplasm surrounding the nucleus.
• Normal erythroblasts:

The early erythroblast is similar to a proerythroblast but is smaller and no longer has visible nucleoli.

The intermediate erythroblast and the late erythroblast show a progressive reduction in cell size, reduction in cytoplasmic basophilia and increase in chromatin clumping.The cytoplasm of the late erythroblast may have a pink tinge attributable to haemoglobin.

• Myeloblast: with a high nucleocytoplasmic ratio, diffuse chromatin pattern and nucleolus.
• Promyelocyte: larger and has a lower nucleocytoplasmic ratio and abundant azurophilic granules
• Myelocytes: are smaller than promyelocytes and have specific granules that indicate whether they are of neutrophil, eosinophil or basophil lineage. The nucleolus is no longer visible.
• Metamyelocyte: differs from a myelocyte in having some indentation of the nucleus.
• Band or juvenile Neutrophils: There are smaller numbers of cells of neutrophil lineage with non-segmented nuclei. They are referred to as neutrophil band cells or band forms. They are less mature than segmented neutrophils.
• Megakaryoblasts: are the precursors of the megakarycytes. They may show cytoplasmic blebbing.
• Megakaryocytes: are large cells which can be identified with low power. Their numbers are very variable in normal bone marrow films, being partly related to the number of fragments present.
• Grading of iron stores on the bone marrow aspiration:

0 : no iron granules seen

1: Small granules in reticulum cells only under oil-immersion

2: Few small granules visible with low power lens

3: Numerous small granules in all marrow particles

4: large granules in small clumps

5: Dense large clumps of granules

6: very large deposits obscuring the marrow cells

Grade 0: iron deficiency (a minimum of 7 BM particles must be available before concluding that hemosiderin is absent)

Grade 1: Diminished iron stores

Grade 2 & 3: normal iron stores

Grade 4 & 6: Increased iron stores

Quality control procedure:

• All the bone marrow aspiration cases are reported by a panel of three pathologists
• Inter laboratory comparison is done once in every 6 month with NABL accredited laboratory (Metropolis lab)

Calculation of results and uncertainty:

Not applicable

Biological reference interval:

• M:E ratio: 2.4 (1.3-4.6)
• Myeloblasts: 1.4 (0-3)
• Promyelocytes: 7.8 (3.2-12.4)
• Myelocytes: 7.6 (1.9-13.3)
• Metamyelocytes: 4.1 (2.3-5.9)
• Neutrophils plus band cells: 34.2 (23.4-45)
• Eosinophils: 2.2 (0.3-4.2)
• Basophils: 0.1 (0-0.4)
• Monocytes: 1.3 (0-2.6)
• Erythroblasts: 25.9 (13.6-38.2)
• Lymphocytes: 13.1 (6-20)
• Plasma cells: 0.6 (0-1.2)

Reportable interval for examination results:

Not applicable

Critical values:

• Aplastic Anemia
• Acute leukemia
• Other malignancy

Interpretation by the laboratory:

The marrow report includes an estimate of cellularity, an estimate of the number of megakaryocytes, the M:E ratio, statement about any cytologic or maturation abnormalities, an estimate of the storage iron and proportion of sideroblasts, and statements about any other abnormal findings present

Potential sources of variability:

• Bone marrow cellularity is better assessed on biopsy, since in aspiration there is an element of dilution
• Stromal changes like reticulin fibrosis, amyloidosis, gelatinous marrow transformation is better diagnosed on marrow trephine biopsy as cellularity in above mentioned condition varies with the type of lesions and severity
• Diseases and neoplasms which infiltrate the marrow in focal or nodular pattern and/or associated with increased marrow reticulin are difficult to aspirate and are diagnosed on biopsy only, e.g. Hodgkin’s deposits in the marrow, tubercular granulomas and metastatic deposits.