processing of bone marrow trephine biopsy

there is no standard method for processing of bone marrow trephine biopsies. there are various fixatives and decalcifying agents . depending upon need of IHC and cytogenetics, we can decide

1. PROCESSING OF BONE MARROW TREPHINE BIOPSY Dr. K anwalpreet Kaur

2. BONE MARROW EXAMINATION Consists of BONE MARROW ASPIRATION: cell morphology and enumeration of marrow cellular elements BONE MARROW BIOPSY: cellularity, fibrosis, marrow architecture or vasculature, infections or infiltrative disorders

3. INDICATIONS 1) Unexplained anaemia, abnormal red cell indices, cytopenia or cytoses 2) Investigation of abnormal peripheral blood smear morphology e.g. leukoerythroblastic picture suggestive of bone marrow pathology 3) 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

4. INDICATIONS 5) Unexplained focal bony lesions on radiography 6 ) Diagnosis, staging and follow up of small round cell tumours of childhood 7) Pyrexia of unknown origin or specific infections e.g. miliary tuberculosis, leishmaniasis , malaria 8) Evaluation of iron stores 9) Investigation of lipid/glycogen storage disorders 10) Exclusion of haematological disease in potential allogenic stem cell transplant donors

5. 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.

6. 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

7. 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.

8. Bone marrow aspiration needle Salah klima islam

9. BONE MARROW BIOPSY NEEDLE: JAMSHIDI NEEDLE A: HOLLOW NEEDLE WITH BEVELED TIP B: OBTURATOR / STYLET C: PROBE TO EXPRESS BIOPSY FROM NEEDLE (probe)

10. BONE MARROW BIOPSY NEEDLE: JAMSHIDI NEEDLE

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

12. 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

13. Ideal core : gross

14. Procedural haemorrahage

15. 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

16. COLLECTION OF BONE MARROW TREPHINE SPECIMENS FIXATION DECALCIFICATION PROCESSING STAINING MICROSCOPY SUPPLEMENTARY INVESTIGATIONS

17. COLLECTION OF BONE MARROW TREPHINE SPECIMENS

18. FIXATION Various fixatives – 1) neutral buffered formalin -6hrs 2) zinc formaldehyde 3) B5 (mercuric chloride, sodium acetate and formalin) 4) AZF ( acetic acid-zinc-formalin)- 6hrs 5) IBF (isotonic buffered formalin) 6) B ouin’s fixative ( picric acid, acetic acid and formaldehyde)- 4-12 hrs 7) Zenkers fixative(mercuric chloride, potassium dichromate,sodium sulfate and glacial acetic acid) -4h

19. 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.

20. 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

21. 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

22. 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.

23. 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

24.  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

25. 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

26. 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

27. AZF : preparation zinc chloride – 12.5g Glacial acetic acid- 7.5ml Concentrated formaldehyde 150ml Distilled water 1000ml Hematologist is instructed to place the freshly obtained BMT specimens directly into AZF fixative and transport it

28. 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

29. 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

30. WHAT WE FOLLOW AT SMS BIOPSY RECEIVED IN FORMALIN BIOPSY IN BOUINS’ X 2HRS BIOPSY IN 5% NITRIC ACID X 2HRS

31. Biopsy is then kept in water overnight and next day in running water for 3-4 hrs and then is paraffin processed

32. 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

33. Standard sections To ensure adequate view of tissue , sections are taken from three levels:25%,50% and 75% into cross sectional diameter of core

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

35. 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

36. 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

37. 2) RETICULIN STAIN – silver impregnation method ( gomori method and gorden and sweets method) 3) Prussion blue ( perls ’) stain – iron

38. M ost 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

39. Normal bone marrow biopsy

40. Different areas of bone marrow biopsy

41. 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

42. Thank you….