PROCESSING OF BONE MARROW TREPHINE BIOPSY

Dr. Kanwalpreet Kaur

BONE MARROW EXAMINATION

Consists of 1) BONE MARROW ASPIRATION: cell

morphology and enumeration of marrow cellular elements

2) BONE MARROW BIOPSY: cellularity, fibrosis, marrow architecture or vasculature, infections or infiltrative disorders

INDICATIONS 1) Unexplained anaemia, abnormal red cell indices, cytopenia or cytoses2) Investigation of abnormal peripheral blood smear morphology e.g. leukoerythroblastic picture suggestive of bone marrow pathology3) Diagnosis, staging and follow up of malignant haematological disorders ( acute and chronic leukemias, myelodysplastic syndromes, chronic myeloproliferative disorders, lymphomas, plasma cell myeloma, amyloidosis, mastocytosis)4) Investigation of suspected bone marrow metastasis

INDICATIONS5) Unexplained focal bony lesions on radiography6) Diagnosis, staging and follow up of small round cell tumours of childhood7) Pyrexia of unknown origin or specific infections e.g. miliary tuberculosis, leishmaniasis, malaria8) Evaluation of iron stores9) Investigation of lipid/glycogen storage disorders10) Exclusion of haematological disease in potential allogenic stem cell transplant donors

CONTRAINDICATIONS Sternal aspirate is absolutely contraindicated in

patients with diseases associated with marked osteoporosis including multiple myeloma

Coagulopathies : BMB is contraindicated Isolated thrombocytopenia is not a

contraindication . Adequate post procedure supervision is must.

SITE POSTERIOR ILIAC CREST- most common

site ANTERIOR ILIAC CREST- in cases where

previous radiation, surgery or discomfort

STERNUM AT SECOND INTERCOSTAL SPACE –don’t allow biopsies

Either the aspirate or biopsy can be performed first. If aspirate is performed first, trephine biopsy should be

performed through same incision, approximately 0.5-1 cm away from the site of aspiration to avoid damaged or haemmorhagic trephine biopsy.

Bone marrow aspiration needle

Salah klima islam

BONE MARROW BIOPSY NEEDLE: JAMSHIDI NEEDLE

A: HOLLOW NEEDLE WITH BEVELED TIPB: OBTURATOR / STYLET C: PROBE TO EXPRESS BIOPSY FROM NEEDLE (probe)

BONE MARROW BIOPSY NEEDLE: JAMSHIDI NEEDLE

PROCEDURE

https://www.youtube.com/watch?v=NkdsLHBCreI

IDEAL CORE Core of atleast 1.5cm – atleast three intact

intertrabecular marrow spaces- free of artefacts Biopsy specimen shrinks by 20% after processing for focal pathologies (lymphoma infiltration /

metastatic deposits)- bilateral trephine biopsies may be obtained ; totalling length of 20-30 mm

Ideal core : gross

Procedural haemorrahage

SOURCES OF ERROR IN INTERPRETING BMT HISTOLOGY- preanalytical errors

Inadequate clinical, hematological, genetic and radiological information

Inadequate specimen- too small, too crushed, both, poorly decalcified

Inadequate sections- thickness, number of levels Inadequate stains- poor technical quality, limited stains

COLLECTION OF BONE MARROW TREPHINE SPECIMENS

FIXATION DECALCIFICATION PROCESSING STAINING MICROSCOPY SUPPLEMENTARY INVESTIGATIONS

COLLECTION OF BONE MARROW TREPHINE SPECIMENS

FIXATION Various fixatives –1) neutral buffered formalin -6hrs2) zinc formaldehyde3) B5 (mercuric chloride, sodium acetate and formalin)4) AZF ( acetic acid-zinc-formalin)- 6hrs5) IBF (isotonic buffered formalin)6) Bouin’s fixative ( picric acid, acetic acid and formaldehyde)- 4-12 hrs7) Zenkers fixative(mercuric chloride, potassium dichromate,sodium sulfate and glacial acetic acid) -4h

Fixation times varies from 1h to maximum of >24h depending upon the fixative used

Trephine biopsy cores should be fixed in formol-saline for a minimum of 18 h but fixation for longer periods does not affect subsequent processing or morphology and is desirable for large samples

Mercurial fixatives such as B5 and Zenker’s fixative result in improved morphology over formalin fixation

The reactivity of antibodies used for immunohistochemical staining may also be affected by the choice of fixative and Zenker’s fixative can destroy chloroacetate esterase activity.

DECALCIFICATION EDTA(5%) Formic acid(5%) Acetic acid Picric acid Nitric acid (5%) Commercial decalcifying agents

Decalcification chelates storage iron,affects morphology and cytological details, ability to perform histochemistry and IHC and to retrieve material suitable for molecular analysis

Decalcification time varies from 15 min to 72 hours depending on decalcifying agent and size of biopsy Decalcification with EDTA is slower(24-48 hrs) than

other agents but result in better preservation of nucleic acids

Warm incubation with agitation or microwave heating can be employed to increase efficiency and reduce time required for decalcification

Weak acids like aqueous 10% formic acid are also slow in action (2-3 days) and also cause tissue distortion

Strong acids like fresh aqueous nitric acid 5% are rapid decalcifiers

Using inorganic acids, such as hydrochloric or nitric acid, should be avoided as this affects morphological preservation adversely and impairs metachromatic staining of sections, e.g. with Giemsa or toluidine blue.

There is NO SINGLE BEST METHOD

Fixation and decalcification schedules varies widely, depending on local priorities for speed, IHC and preservation of nucleic acids for molecular studies

Priniciple- to ensure proper fixation prior to exposure of tissue to acidic or chelating decalcifying agents

 International Council for Standardization in Hematology (ICSH) recommends neutral buffered formalin with EDTA decalcification - adequate pathology , preserves antigens for IHC and nucleic acids for molecular studies

HAMMERSMITH PROTOCOL specimens are transported and fixed in acetic acid-zinc-

formalin fixative for overnight decalcified in 10% formic acid-5%formaldehyde processed in paraffin wax embedding

Wilkins et al. also recommends use of AZF for 6hrs. Addition of zinc in fixative stabilize nucleic acids, can protect

against hydrolysis in presence of weak acid

AZF fixative results in superior antigen preservation, well preserved RNA and DNA for FISH and molecular analysis

Hence, AZF preparation can effectively speed decalcification without detriment to morphology or antigen preservation

AZF : preparation zinc chloride – 12.5gGlacial acetic acid- 7.5mlConcentrated formaldehyde 150mlDistilled water 1000ml

Hematologist is instructed to place the freshly obtained BMT specimens directly into AZF fixative and transport it

FIXATION - in AZF overnight; next day biopsy is washed with distilled water for 30 min

DECALCIFICATION- 10% formic acid and 5% formaldehyde – decalcify in 6hrs

Processing and embedding in paraffin wax Sectioning and staining

Turnout time PROCEDURE TIME(HRS) PROCEDURE

COMPLETION ON DAY

FIXATION 20-24 hrs 1

DECALCIFICATION 6 1

PROCESSING AND EMBEDDING

12-16 2

SECTIONING FOR H&E 1 2

Paraffin PROCESSING OF TREPHINE BIOPSY SPECIMEN

After decalcification biopsy specimen is embedded in paraffin wax and sections cut on microtome

Recommended thickness 2-3 microns To allow high quality morphological assessment

including cytological evaluation using oil immersion lens

For DNA extraction two 15 micron thick sections are cut

Standard sections To ensure adequate view of tissue , sections are

taken from three levels:25%,50% and 75% into cross sectional diameter of core

Spare sections on poly-l-lysine coated slides are retained between levels 2 and 3 - suitable for use IHC

PLASTIC EMBEDDING

Some laboratories embed trephine cores in plastic resins such as glycol methacrylate or methyl methacrylate without decalcification .

here section cutting requires glass knives or tungsten carbide knives

It gives good cytological details but is technically more difficult and limits the range of immunohistochemical studies and FISH

It may be useful for the evaluation of metabolic bone diseases and histochemical stains ablated by decalcification process

STAINING OF SECTIONS Hematoxylin and eosin Additional- 1) GEIMSA may be done; helpful in identifying plasma cells, mast cells, lymphoid cells and eosinophils and for distinguishing between myeloblasts and proerythroblasts

2) RETICULIN STAIN – silver impregnation method (gomori method and gorden and sweets method)

3) Prussion blue (perls’) stain – iron

Most enzyme histochemistry is unsuccessful because of irreversible denaturation of the enzymes during decalcification and processing.

One exception is acid phosphatase activity which is sometimes retained. When hairy cell leukaemia is suspected, demonstration of tartrate-resistant acid phosphatase (TRAP) activity may be useful

Normal bone marrow biopsy

Different areas of bone marrow biopsy

HISTOLOGY - adequacy and macroscopic appearance of core

• percentage and pattern of cellularity• Bone architecture• Location, number, morphology and pattern of

differentiation for erythroid, myeloid and megakaryocytic lineages,lymphoid cells, plasma cells and macrophages

• Abnormal cells and/ or infiltrates• Reticulin stain • Immunohistochemistry• Histochemistry• Other investigations- FISH, PCR• CONCLUSION

Thank you….