Purpose of examination: Bone marrow biopsy allows histological assessment of cellularity, fibrosis, infections, infiltrative diseases and disease of bone and its cells. Trephine biopsy is essential for diagnosis when dry Tap or blood tap due to fibrotic or densely cellular marrow. It is particularly useful in investigating suspected aplastic or hypoplastic anemia, lymphoma, metastatic carcinoma, myeloproliferative disorder and disease of bone.

Principle of examination: bone marrow biopsy is taken by bone marrow biopsy needle; are fixed in 10% neutral buffered formalin, then decalcified using weak organic acid and then processed as routine histopathology tissue, stained with H & E stain and studied microscopically.

Performance specification: Not applicable.

Sample type required:

Biopsy material processed and stained with H & E stain

Preservative needed:

Neutral buffered formalin

Reagents required:

Formalin

5% HNO3 (decalcifier)

Alcohol

Xylene

Paraffin wax

H & E stain

DPX

Calibration method:

Not applicable

Detailed work bench instruction:

Sites of Bone marrow biopsy:

Posterior superior iliac spine

Anterior superior iliac spine

Procedure:

• Written consent of patient is taken after explanation of procedure and possible risk. Patient is given inj.atropine 0.5ml intra muscular before 15 min of commencement of procedure.
• Patient is asked to take Lateral supine position (knee- chest)
• Bony prominence is felt and site is sterilized with spirit- betadine- spirit.
• Local anesthesia is given with 2ml Lignocaine injection sub-periostealy.
• Bone marrow biopsy needle is inserted perpendicular to the bony prominence by screwing motion. When give away sensation is felt trochar is removed and biopsy needle is inserted exactly 2 cm more deeper.
• Detachment of the bone is done by rotating the needle 360’ and moving vigorously upward- downward and sideways. Then the needle is pulled out.
• The biopsy is taken out with the help of stillete inserting from the tip of needle to prevent compression of biopsy
• Imprint smear (minimum four in no) is taken by rolling the biopsy on slide
• Then the biopsy is placed inside 10% neutral buffered formalin for minimum 6 hrs
• Then it is decalcified with 5% HNO3 (prepared in 10% buffered formalin) for four hours
• Biopsy tissue is processed as per routine histopathology tissue processing. Section of biopsy tissue is should be of 3-4 micron thickness and the stained with H& E stain

Quality control procedure:

• Adequacy of biopsy tissue should be checked:

• tissue should be at least 1.5cm in length; if it is less than 1.5 cm then contra lateral biopsy should be taken
• If biopsy is <1.6cm in length; it is considered adequate if it is positive for granuloma, metastasis, fungus, parasite or lymphoma
• More than 5 inter trabecular spaces should be seen

• Internal Quality Control between 2 observor is done two monthly and records of which are kept in HI:C\Records\file\9\internal quality control records L

Calculation of results and uncertainty:

Not applicable

Biological reference interval:

• Cellularity: assessed by Marrow: fat ratio ( trabeculae should be excluded)

• varies with age; generally assessed as (100 – Age)
• Bone marrow remains only 20-25% cellular at the age of 70 yrs; so gradual decrease in marrow cellularity is expected with increase in age of patient

• Normal erythropoeisis
• Normal Leukopoesis
• Normal megakaryopoeisis

Reportable interval for examination results:

Not applicable

Critical values:

• Aplastic Anemia
• Acute leukemia
• Other malignancy

Interpretation by the laboratory:

Scanner: cellularity; trabeculae

Low power: topography of cells

High power: morphology of cells

Oil immersion: parasite

Potential sources of variability: In adults subcortical marrow is hypoplastic upto a depth of 1 cm and this is a normal variation and therefore such a biopsy should not be reported as hypoplastic/ aplastic anemia. Therefore an adequate biopsy of 2-2.5cm is essential to diagnose aplastic anemia