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 Contraindication
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 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):
Trewhitt KG. Bone marrow aspiration and biopsy: collection and interpretation. Oncol Nurs Forum. Oct 2001;28(9): ;

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. Hemorrhagic Biopsy
Crushed Biopsy

12. Post OP care Adverse events 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?
Unilateral or Bilateral procedure?

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
Smear Preparation
Anti-coagulated sample
Clot preparation
Flow Cytometry (EDTA )
Cytogenetic studies (Heparin)
Molecular studies (EDTA)
Morphology
Cytochemistry
FISH (if required)
Processed as biopsy

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. Properly dried smear
Lysed RBC due to improper drying

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

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

22. BM Biopsy Fixation 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.
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;59:903–911.
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.

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 Embedding 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. H&E
Retic
H&E
LCA

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.

28. Thanks