processing of bone marrow trephine biopsy
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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….
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