Presentation on theme: "Bone Marrow Procedure and Processing"— Presentation transcript:
1Bone Marrow Procedure and Processing Dr Khaliqur RahmanSenior Resident, Hematopathology LaboratoryDepartment of Pathology, TMH
2HistoryInitially 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
3Indications Contraindication Pyrexia of unknown origin(Tuberculosis, leishmaniasis)PancytopeniaThrombocytopeniaRefractory anaemiaStorage diseasesLeukaemiaLeukoerythroblastic picture in peripheral blood.Paraproteinemias (rule out Myeloma)Staging of neoplasm including lymphomaFor stem cell transplantationsContraindication
4Aspiration versus Trephine Complementary AdvantagesBone 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):Trewhitt KG. Bone marrow aspiration and biopsy: collection and interpretation. Oncol Nurs Forum. Oct 2001;28(9): ;
5Focal involvement in a case of neuroblastoma highlighted by synaptophysin
6Focal Paratrabecular aggregate of lymphoma cells in a case of Follicular LymhpomaFocal Paratrabecular aggregate of lymphoma cells, highlighted by cyclin D1 in a mantle cell lymphoma
7Site 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
12Post OP care Adverse events Firm pressure on the aspiration site. If haemorrhage persists, place the patient in the supine positionAnalgesics to alleviate the pain.Adverse events
13Unilateral or Bilateral procedure? Aspirate first or the biopsy first?Unilateral or Bilateral procedure?
14Adequacy 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.
15Processing of BM aspirate Bone Marrow AspirateSmear PreparationAnti-coagulated sampleClot preparationFlow Cytometry (EDTA )Cytogenetic studies (Heparin)Molecular studies (EDTA)MorphologyCytochemistryFISH (if required)Processed as biopsy
16Smears preparation Smears should prepared rapidly Smears should be well spreadSquash or imprint can be prepared as necessary.Sufficient number of slides should be prepared.Smears should be thoroughly air driedA minimum of Romanowsky and Perl’s stain should be doneBain BJ. Bone marrow aspiration. J Clin Pathol 2001;54:657–663.
19Properly dried smearLysed RBC due to improper drying
20Wright’s StainNon Specific esteraseMyeloperoxidasePerl’s Stain
21Processing of BM Biopsy Bone Marrow biopsyFixationImprint SmearDecalcification&Paraffin embeddingMorphological Correlation( Better representation of marrow)SectionsH&E, Special stains Immunohistochemistry
22BM Biopsy Fixation Fixatives Minimum duration of fixation 10% neutral buffered saline18 hrs (up to 48 hrs)Bouins#4-12 hrsZenkers$4hrsB5$4 hrs, not more than 6 hrsAceto-zinc-Formalin(AZF)*Overnight*Hammersmith protocol, $ Mercury based fixatives, #contains picric acidBouins and merrcury based fixatives are good for morphology but IHC is compromisedAZF is better over all fixatives in terms of preservation of morphology, IHC, DNA and RNA.Bain BJ, Clark DM and Wilkins BS. Bone marrow Pathology. 4th edition. pp 601K 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.
23Hammersmith 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 hrSpecimen embedded in paraffin waxThin sections of 1μmThe 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
25Sections 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.
27Take Home MessageBM 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.