Bone Marrow Procedure and Processing

Dr Khaliqur Rahman Senior Resident, Hematopathology Laboratory Department of Pathology, TMH

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

Aspiration versus Trephine

Complementary
Advantages Bone marrow aspiration Fine cytological details, Wider range of cytochemical stains can be used, Ideal for microbiological culture, flow cytometry, cytogenetic and molecular studies. Complete assessment of cellularity and architecture. Detect focal lesions. Useful for assessment of aplastic anemia, metastasis etc. Archival material.

Bone marrow Biopsy

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 involvement in a case of neuroblastoma highlighted by synaptophysin

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

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

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

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

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.

Processing of BM aspirate
Bone Marrow Aspirate

Smear Preparation

Anti-coagulated sample

Clot preparation

Morphology
Cytochemistry FISH (if required)

Flow Cytometry (EDTA )

Processed as biopsy

Cytogenetic studies (Heparin)

Molecular studies (EDTA)

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

Bain BJ. Bone marrow aspiration. J Clin Pathol 2001;54:657663.

Properly dried smear

Lysed RBC due to improper drying

Wrights Stain Myeloperoxidase

Non Specific esterase Perls Stain

Processing of BM Biopsy
Bone Marrow biopsy Fixation Imprint Smear

Decalcification & Paraffin embedding Sections H&E, Special stains

Morphological Correlation ( Better representation of marrow)

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

Take Home Message

BM examination is an invaluable tool in work up of hematolymphoid neoplasm, PUO etc. Its 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.