Bone MarrowProcedure and Processing

Dr Khaliqur Rahman Senior Resident, Hematopathology LaboratoryDepartment 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):14-9www.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 TrephineComplementary

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.

Bone marrow Biopsy Complete 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;

Focal involvement in a case of neuroblastoma highlighted by synaptophysin

Focal Paratrabecular aggregate of lymphoma cellsin 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

Crushed Biopsy

Hemorrhagic 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

Unilateral or Bilateral procedure?

Aspirate first or the biopsy first?

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

Anti-coagulated sampleSmear Preparation Clot preparation

Processed as biopsyFlow Cytometry (EDTA )

Cytogenetic studies (Heparin)

Molecular studies (EDTA)

Morphology

Cytochemistry

FISH (if required)

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.

Lysed RBC due to improper drying

Properly dried smear

Wright’s Stain

Myeloperoxidase Perl’s Stain

Non Specific esterase

Processing of BM BiopsyBone Marrow biopsy

Fixation Imprint Smear

Morphological Correlation ( Better representation of marrow)

Decalcification&

Paraffin embedding

H&E, Special stains Immunohistochemistry

Sections

BM Biopsy Fixation

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.

Fixatives Minimum duration of fixation

10% neutral buffered saline 18 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.

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

Decalcification

• 10%NITRIC ACID• HYDROCHLORIC ACID (HCL)• FORMIC ACID• EDTA

Embedding•Paraffin

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.

LCA

H&E

H&E Retic

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.

Thanks