Trephine biopsy staining

For patient safety and convenience, biopsies are usually performed on the posterior iliac crest. The biopsy specimen should measure at least 1.6 cm and, if it does not, consideration should be given to repeating the procedure, possibly on the contralateral iliac crest. If bone marrow aspiration is found to be impossible, imprints from the biopsy specimen should be obtained. Otherwise, the specimen is placed immediately into fixative and after fixation is embedded in a resin or, more usually, decalcified and embedded in paraffin wax. Thin sections are cut and are stained, as a minimum, with haematoxylin and eosin and with a reticulin stain

Indications for performing a trephine biopsy. It can be:

+ Inadequate or failed aspirate.

+ Need for accurate assessment of cellularity, whether increased or decreased.

+ Suspected focal lesion (for example, suspected granulomatous disease or lymphoma).

+ Suspected bone marrow fibrosis.

+ Need to study bone marrow architecture.

+ Need to study bone structure or bone marrow blood vessels

Site and technique of biopsy

A trephine biopsy is usually most easily carried out on the posterior superior iliac spine, with the patient in the left or right lateral position and with the knees drawn up.

Needles: Jamshidi needle (8-11 size)

1% lidocaine (3ml) Anesthesia

A) Procedure

Appropriate sterile gloves should be worn and anaseptic technique must be used.A core of bone is cut with a hollow needle and the specimen is then aspirated into a syringe; marrow structure is preserved. The first step is to identify the parts of the sampling site.

1. Mark the preferred sampling site.

2. Administer anaesthesia as described for needle aspiration. When the trephine needle is assembled, it is inserted into the sampling site either directly, or after making a small (0.2cm) incision to facilitate access to the bone. The needle is pushed into the bone by rotating it on its axis.

3. When the desired depth is reached, the stylet is withdrawn and the needle is advanced using the same rotating movement.

4. The depth of the needle is measured using a stylet. A specimen measuring approximately 2cm in length should be collected.

5. When the correct depth has been ascertained, the needle is rotated in the opposite direction to help loosen the specimen. The needle, with the specimen, is then withdrawn using a rotational movement.

6. Then, the piece of bone sample will be transferred to formalin container for preservation

B) Fixation:

1) Neutral buffered 10% formalin (6 hrs)

Once received in the laboratory, the BMT specimens are left to fix in formalin overnight. The next morning (after 20–24 h), the biopsy specimen is washed in distilled water for 30 min. Fixative is used for the preservation of the specimen or bone marrow with bone (Biopsy) to retain its morphology and for transportation purpose.

C) Decalcification:

1) 5% EDTA

2) 5% Formic acid

The biopsy specimens are left to decalcify for about 6 hrs-24 hrsbefore being processed and embedded in paraffin wax. It is to remove calcium salt from the tissue and to make it soft for cutting in a microtome.

E) Tissue sectioning

Sections, 1‐μm thick (microtome set for 1 μm sections), are cut from the paraffin‐wax blocks with the routine rotary microtomes in the laboratory.

D)Staining

The sections are stained with H&E, Giemsa, Perl's (iron) and reticulin (silver) stains.

1) H&E Stain (Haematoxylin and eosin stain):

This stain is performed to demonstrate the general structure of tissue. It is a routine stain. 

Principle: Haematoxylin dyes are basic dyes, hence they are positively charged.  Therefore it will stain the acidic structures of tissues and cell structures (basophilic), purplish-blue.Eosin dye is acidic dye hence it as a negative charge (eosinophilic). Therefore it stains the basic structures of a cell (acidophils), giving them a red or pink color, for example, the cytoplasm is positively charged, and therefore it will take up the eosin dye, and appear pink.

Reagents:

1. Haematoxylin solution

2. 1% Acid alcohol

3. 1% aqua. Eosin solution

Procedure:

1. Take the section down to water (To De-wax).

2. Stain with Haematoxylin with 5-10 minutes.

3. Rinse in water for few seconds.

4. Differentiate in 1% acid alcohol for 10-15 sec.

5. Wash under running tap water for 5 minutes.

6. Stain with 1% eosin solution f or 5 minutes.

7. Wash under running tap water for 30 sec

8. Dehydrate, clear and mount.

Results:

Nuclei: Bright blue

Cytoplasm: pale pink

Muscle, Keratin, colloid: Bright pin

Erythrocytes: Orange-red

A. Megakaryocytes   B. Myeloma, leukemia

Clear area is fat