BM Procedure and Processing
BM Procedure and Processing
BM Procedure and Processing
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 www.madametalbot.com, www.tuesday-johnson.tblr.com
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
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;
Focal Paratrabecular aggregate of lymphoma cells, highlighted by cyclin D1 in a mantle cell lymphoma
Needles used
Single use Needle
Reusable needle
Hemorrhagic Biopsy
Crushed Biopsy
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
Processing of BM aspirate
Bone Marrow Aspirate
Smear Preparation
Anti-coagulated sample
Clot preparation
Morphology
Cytochemistry FISH (if required)
Processed as biopsy
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 Perls stain should be done
Processing of BM Biopsy
Bone Marrow biopsy Fixation Imprint Smear
Immunohistochemistry
BM Biopsy Fixation
Fixatives 10% neutral buffered saline Bouins# Zenkers$ B5$ Aceto-zinc-Formalin(AZF)* Minimum duration of fixation 18 hrs (up to 48 hrs) 4-12 hrs 4hrs 4 hrs, not more than 6 hrs 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.
Bain BJ, Clark DM and Wilkins BS. Bone marrow Pathology. 4th 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;59:903911. Bonds LA, Barnes P, Foucar K, et al. Acetic acid-zinc-formalin: a safe alternative to B-5 fixative. Am J Clin Pathol 2005;124:20511.
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 2024 h), specimen is washed in distilled water for 30 min. Gooding and Stewarts decalcification fluid (10% formic acid and 5% formaldehyde)- 6 hr Specimen embedded in paraffin wax Thin sections of 1m The sections are stained with, H&E, Giemsa, Perls (iron) and reticulin (silver) stains. Additional unstained sections are placed on poly-L-lysine coated slides for immunostaining as necessary
Decalcification
10%NITRIC ACID HYDROCHLORIC ACID (HCL) FORMIC ACID EDTA
Embedding
Paraffin
Sections
Thin sections, not more than 3m. 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.
H&E
Retic
H&E
LCA