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Bone Marrow Procedure and Processing Dr Khaliqur Rahman Senior Resident, Hematopathology Laboratory Department of Pathology, TMH.

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Presentation on theme: "Bone Marrow Procedure and Processing Dr Khaliqur Rahman Senior Resident, Hematopathology Laboratory Department of Pathology, TMH."— Presentation transcript:

1 Bone Marrow Procedure and Processing Dr Khaliqur Rahman Senior Resident, Hematopathology Laboratory Department of Pathology, TMH

2 History Initially entailed the drilling of cranial bones as a form of medical intervention for headaches and mental illnesses. However it was not until 1905, when the Italian physician Pianese reported bone marrow infiltration by the parasite Leishmania, that this procedure was applied toward clinical evaluation. Parapia LA. Trepanning or trephines: a history of bone marrow biopsy. Br J Haematol. Oct 2007;139(1):14-9,

3 Indications Pyrexia of unknown origin(Tuberculosis, leishmaniasis) Pancytopenia Thrombocytopenia Refractory anaemia Storage diseases Leukaemia Leukoerythroblastic picture in peripheral blood. Paraproteinemias (rule out Myeloma) Staging of neoplasm including lymphoma For stem cell transplantations Contraindication

4 Aspiration versus Trephine Complementary Advantages Bone marrow aspirationFine cytological details, Wider range of cytochemical stains can be used, Ideal for microbiological culture, flow cytometry, cytogenetic and molecular studies. Bone marrow BiopsyComplete assessment of cellularity and architecture. Detect focal lesions. Useful for assessment of aplastic anemia, metastasis etc. Archival material. Bain BJ. Bone marrow trephine biopsy. J Clin Pathol. Oct 2001;54(10):737-42. Trewhitt KG. Bone marrow aspiration and biopsy: collection and interpretation. Oncol Nurs Forum. Oct 2001;28(9):1409-15;

5 Focal involvement in a case of neuroblastoma highlighted by synaptophysin

6 Focal Paratrabecular aggregate of lymphoma cells in a case of Follicular Lymhpoma Focal Paratrabecular aggregate of lymphoma cells, highlighted by cyclin D1 in a mantle cell lymphoma

7 Site for aspiration. Posterior superior iliac spine Anterior superior iliac crest. Spinous process of the lumbar vertebrae. The sternum. The tibia is sampled only for infants younger than 1 year

8 Needles used Single use Needle Reusable needle



11 Crushed Biopsy Hemorrhagic Biopsy

12 Post OP care Firm pressure on the aspiration site. If haemorrhage persists, place the patient in the supine position Analgesics to alleviate the pain. Adverse events

13 Unilateral or Bilateral procedure? Aspirate first or the biopsy first?

14 Adequacy of the specimen For a bone marrow biopsy, the accepted norm has been a length of 1.5cm, 5-6 inter-trabecular spaces and absence of handling or processing artifacts.

15 Processing of BM aspirate Bone Marrow Aspirate Anti-coagulated sampleSmear PreparationClot preparation Processed as biopsy Flow Cytometry (EDTA ) Cytogenetic studies (Heparin) Molecular studies (EDTA) Morphology Cytochemistry FISH (if required)

16 Smears preparation Smears should prepared rapidly Smears should be well spread Squash or imprint can be prepared as necessary. Sufficient number of slides should be prepared. Smears should be thoroughly air dried A minimum of Romanowsky and Perl’s stain should be done Bain BJ. Bone marrow aspiration. J Clin Pathol 2001;54:657–663.



19 Lysed RBC due to improper drying Properly dried smear

20 Wright’s Stain Myeloperoxidase Perl’s Stain Non Specific esterase

21 Processing of BM Biopsy Bone Marrow biopsy Fixation Imprint Smear Morphological Correlation ( Better representation of marrow) Decalcification & Paraffin embedding H&E, Special stains Immunohistochemistry Sections

22 BM Biopsy Fixation Bain BJ, Clark DM and Wilkins BS. Bone marrow Pathology. 4 th edition. pp 601 K N Naresh, I Lampert, R Hasserjian, D Lykidis. Optimal processing of bone marrow trephine biopsy: the Hammersmith Protocol. J Clin Pathol 2006;59:903–911. Bonds LA, Barnes P, Foucar K, et al. Acetic acid-zinc-formalin: a safe alternative to B-5 fixative. Am J Clin Pathol 2005;124:205–11. FixativesMinimum duration of fixation 10% neutral buffered saline18 hrs (up to 48 hrs) Bouins # 4-12 hrs Zenkers $ 4hrs B5 $ 4 hrs, not more than 6 hrs Aceto-zinc-Formalin(AZF)*Overnight *Hammersmith protocol, $ Mercury based fixatives, # contains picric acid Bouins and merrcury based fixatives are good for morphology but IHC is compromised AZF is better over all fixatives in terms of preservation of morphology, IHC, DNA and RNA.

23 Hammersmith Protocol. Fix in AZF [zinc chloride, 12.5 g; concentrated formaldehyde,150 ml; glacial acetic acid, 7.5 ml; and distilled water, to1000 ml] overnight. The next morning (after 20–24 h), specimen is washed in distilled water for 30 min. Gooding and Stewart’s decalcification fluid (10% formic acid and 5% formaldehyde)- 6 hr Specimen embedded in paraffin wax Thin sections of 1μm The sections are stained with, H&E, Giemsa, Perl’s (iron) and reticulin (silver) stains. Additional unstained sections are placed on poly-L-lysine coated slides for immunostaining as necessary

24 Decalcification 10%NITRIC ACID HYDROCHLORIC ACID (HCL) FORMIC ACID EDTA Embedding Paraffin

25 Sections Thin sections, not more than 3μm. Serial sections from multiple levels should be examined. A minimum of H&E and reticulin stain is recommended. Additional unstained slides should be cut in advance for IHC stains.

26 LCA H&E Retic

27 Take Home Message BM examination is an invaluable tool in work up of hematolymphoid neoplasm, PUO etc. It’s a painful procedure and sample obtained is precious. Aspiration and biopsy are the two facets of same coin. A good diagnosis rest on good processing.


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