Bone marrow procedures involve bone marrow aspiration and trephine biopsy to examine the bone marrow. Bone marrow aspiration is a simple, safe outpatient procedure that allows examination of individual cells and their morphology. Trephine biopsy provides larger samples for examining marrow structure and architecture, and is valuable for diagnosing conditions with a "dry tap". Proper techniques and precautions are important to perform bone marrow procedures safely and obtain adequate samples for diagnosis.

1. Bone Marrow
Procedures
and
Dr Richards Kakumanu

2. Moderators
Dr V Vijay Sreedhar (Prof & HOD)
Dr Manimekhala (Assoc. Prof)
Upgraded Department of Pathology

3. Marrow Specimen

4. Needle Aspiration

5. Percutaneous
Trephine Biopsy

6. Surgical Biopsy

7. Why do we need to do bone marrow
studies?

8. Bone Marrow Aspiration

9. • Simple and safe
• Repeated many times
• Performed on outpatients
• Safe in almost circumstances

10. Advantages
• Individual cells are well preserved
• Subtle differences between the cells recognized

11. Disadvantages
• Arrangement of cells in the marrow
• Relationship between the cells
• Fibrotic marrows: aspiration of blood

14. General considerations
• Iliac spines: advantages of trephine biopsy
• Obese and immobile patients: technical difficulties
• Sternum should be avoided in children
• Danger of perforating inner cortical layer and
damage to the underlying large blood vessels and
right atrium

15. Important considerations
• Always wear surgical gloves
• Avoid needle stick injuries
• Local and oral analgesia
• Use only needles designed for the purpose

16. Marrow puncture needles
• Needles should be stout
• Hard stainless steel
• About 7-8 cm in length
• Well fitting stilette
• Adjustable guard

17. • Most common reusable needles:
Klima and Salah
• Point of the needle and the edge
of the bevel must be kept well
sharpened

18. • Islam's bone marrow aspiration
needle: the dome-shaped
handle and the T-bar are
intended to provide stability and
control during the operation
• Disposable bone marrow
aspiration needles

19. • Clean the skin
• 70% alcohol
• 0.5% chlorhexidine(5% diluted 1
in 10 in ethanol)

20. • Infiltrate the skin, subcutaneous
tissue, and periosteum
• 2% lignocaine 2-5ml

21. WAIT...

22. • Pass the needle with a boring movement
• Needle - perpendicular into the cavity
• Remove the stilette when bone has been
penetrated
• Attach a 1-2ml syringe
• Aspirate marrow contents
• Second sample: Attach a second 5-10 ml syringe
for cytogenetic and immunophenotypic analysis

23. • As a rule material can be sucked into the syringe
without difficulty
• If unable to aspirate: Insert the stilette, push the
needle and then Aspirate
• Dry tap: failure to Aspirate marrow suggests
fibrosis or infiltration
• Dry tap: insert the stilette, push any material in the
lumen onto a slide
• Obese patients: CT guided marrow sampling

24. • Ethylenediaminetetra-acetic acid(EDTA)
• Preservative free heparin: phenotyping and
cytogenetic analysis
• Preservative in fixative: histopathology
• Fixation in absolute methanol: romanosky method
or perls' stain or cytochemical staining

25. Puncture of the Ilium

26. Puncture of the ilium
• Usual sites: Posterior and anterior iliac spines
• Center of the oval posterior superior iliac spine
• 2cm posterior and 2cm inferior to anterior superior
iliac spine
• Posterior iliac spine: 1) overlies a large marrow
containing area. 2)relatively large volumes of
marrow can be aspirated
• Posture: 1)patient lying sideways or 2)prone

27. Puncture of the
sternum

28. Puncture of the sternum
• Avoid pushing the aspiration needle through the
bone
• Usual sites: 1) manubrium, 2) 1st or 2nd parts of
the body of sternum

29. Manubrium
• Denser bone than body of
sternum
• More fatty marrow in elderly
subjects
• Thickness of cortex: 0.2-5.0mm
• Difficult to ascertain that the
needle point has reached the
cavity of the bone
• Site: about 1cm above the
sternomanubrial angle and
slightly to one side of the
midline

30. Body of Sternum
• Site: opposite the second
intercostal space
• Slightly to one side of the
midline

31. • Essential: Needle with a guard
• Adjust the guard When needle
reaches the periosteum
• Allow it to penetrate about 5mm
further
• Push the needle with a boring
motion, enter the cavity and
Aspirate

32. Puncture of the
Spinous process

33. Puncture of the
Spinous process
• Done in adults
• Spines of the lumbar vertebrae
• Not difficult as bones lie
superficially
• More pressure is required
• Patient sitting up or lying
sideways
• Pass the needle at right angles
to the skin surface
• Into the spine of the Lumbar
vertebra slightly lateral to the

34. Comparison of different sites for marrow
puncture
• In general, the overall cellularity, the hemopoietic
maturation pathways, and the balance between
erythropoiesis and leucopoiesis are similar at all
sites
• Considerable variation in the composition of the
cellular marrow in certain conditions
• Aspiration from only one site may give misleading
information. Eg: aplastic anemia - patchily affected
• Dry tap/bloody tap: advantage in choice of several
sites

35. Aspiration of bone
marrow in children
• Small babies: medial aspect of
upper end of tibia, just below
the level of tibial tubercle
• Caution! Vulnerable to fractures
and laceration of adjacent major
blood vessels
• Children: iliac puncture(posterior
spine)
• Older obese children: anterior
ilium
• Tibial cortical bone: too dense,
marrow normally less active

36. Processing of Aspirated Bone Marrow

37. • Preparing films from Bone Marrow Aspirates
• Concentration of Bone marrow by centrifugation
• Preparation of films of post-mortem bone marrow

38. Processing of marrow Aspirates

39. General considerations
• 0.3 ml of marrow fluid from a single site
• >0.3ml: little advantage - peripheral blood dilution
• A second syringe: 5-10 ml of marrow -
immunophenotyping, cytogenetics and molecular
studies
• Sample of peripheral blood: finger prick or
venepuncture
• Good practice: obtain full blood count and storage

40. Preparing films
from Bone Marrow
Aspirates
• Smears should be made without
delay
• Smear length: 3-5cm
• Glass spreader: smooth edged,
not more than 2cm width
• Marrow fragments dragged
behind the spreader
• Fragments leave a trail of cells
behind them
• Spreading should be towards
the label area.

41. • Insufficient fragments: can be concentrated
• Deliver single drops of Aspirate onto slides about
1cm from one end
• Most of the blood is quickly sucked off from the
edge of the drop using the marrow syringe or a
fine plastic pipette
• Irregularly shaped marrow fragments tend to be
left behind, can be lifted off with a spreader
• Smears can be made

42. • Thorough drying, fix the smears,
and stain (Romanosky)
• A longer fixation time(at least 20
min in methanol) is essential for
high-quality staining

43. • Perls' method: demonstrates the
presence or absence of iron
• Atleast one film should be fixed
for perls' stain
• Overnight drying may be
necessary to achieve to achieve
optimal results

44. • Satisfactory: only when marrow
particles and free marrow cells
can be seen in stained films
• Differential counts should be
made in cellular trails
commencing from the marrow
fragment and working back
towards the head of the film
• Smaller numbers of cells from
the peripheral blood are
included in a differential count

45. • Appropriate amounts of
anticoagulant for the volume of
marrow to be anticoagulated are
used
• Gross excess of anticoagulant:
masses of pink-staining
amorphous material may be
seen
• Clumping of some erythroblasts
and reticulocytes may be seen

46. Concentration of bone marrow by
centrifugation
• To concentrate the marrow cells
• To assess the relative proportions of marrow cells,
peripheral blood and fat in aspirated material
• Useful in poorly cellular samples

47. Preparation of films of post-mortem bone
marrow
• Smears: Seldom satisfactory
• Satisfactory results: procedure must be Carried
out as soon after death as possible
• Majority of cells tend to break up when making
films
• Better preservation: a small piece of marrow is
suspended in 1-2ml of 5% bovine albumin( 1 vol.
30% albumin, 5 vol. 9 g/l NaCl)
• The suspension is then centrifuged

48. Trephine Biopsy

49. • A little less simple
than aspiration
• Can be performed
on outpatients or at
bedside
• Structure of
relatively large
pieces of marrow
• Imprint smears:
morphological

50. • Invaluable in the diagnosis of conditions that yield a
"dry tap".
• Hodgkin's disease, lymphoma: disrupted
architecture of the marrow is an important
diagnostic feature
• Usual site: posterior iliac spine
• Posterior iliac spine: longer, larger samples. Less
comfortable for the patient
• Anterior iliac spine can also be used

51. • Insert the biopsy needle into the
bone
• Obtain a core of tissue using a
to-and-fro rotation
• Main problems: specimen may
be crushed, distortion of the
architecture, difficulty to detach
the core of the bone from inside
the marrow space
• Trephine biopsy needles:
specifically designed to
overcome theses problems

52. Jamshidi trephine
• Tapering end to reduce crush
artefact

54. Islam trephine
• Has a core securing device
• The distal cutting edge is
shaped to hold the core secure
during extraction of the material

55. • Larger specimens: trephine needles with bores of
4-5mm
• Occasionally used needles: 2-mm bore
microtrephine, Vim-Silverman needle
• Smaller yield of marrow specimen that are prone
for fracturing

56. a and neutropenia
in small preterm
neonates
• 19 G, half-inch Osgood needle
• Introduced 2cm below tibial
tuberosity
• The trocar is removed
• The hollow needle is advanced
by twisting 2-3mm into the
marrow space
• Suction applied with a syringe
until marrow appears
• Then needle and syringe are
withdrawn

57. • The marrow clot is gently dislodged with the tip of
a needle and placed into fixative
• The specimen is processed
• Decalcification is not required

58. Complications of bone marrow biopsy

59. • Generally a safe procedure
• Serious adverse events <0.05%
of procedures
• Most common complication:
bleeding
• Gluteal compartment syndrome
• Very rarely death
• Bleeding is related to
impairment of platelet function
than to thrombocytopenia or a
coagulation factor defect

60. Imprints from bone marrow trephine biopsy

61. • Can be taken before the
specimen is transferred into
fixative
• Particularly useful if the bone
marrow Aspirate is inadequate
• Bony core is gently dabbed or
rolled across the slide
• Fixed and stained
• Allows immediate examination
of cells that fall out of the
specimen onto the slide
• May provide a diagnosis several

62. Processing of bone marrow trephine biopsy
specimens

63. • Fixed in 10% formal saline
• Buffered to ph 7, for 12-48 hrs
• Decalcifying, dehydrating and
embedding in paraffin wax
• Cell shrinkage and distortion
from the decalcification process
may distort cellular detail
• Methyl methacrylate(plastic)
embedding

64. Staining of Sections of Bone Marrow
Trephine Biopsy Specimens

65. • Routinely stained for H& E
• Silver impregnation method for reticulin
• Romanosky dyes: MGG - hemopoietic cells may be more easily identified
• Perls' reaction for iron

66. • H&E staining: excellent for demonstrating the
cellularity and pattern of the marrow
• Pathological changes: fibrosis, granulomata,
carcinoma cells
• IHC: paraffin/plastic embedded specimens

67. • Silver impregnation:
stains the
glycoprotein matrix
• Reticulin: an early
form of collagen
• Increase in marrow
reticulin: increase in
the number and
thickness of fibers
• Eg:

68. • In myelofibrosis or
myelosclerosis, a more "mature"
form of collagen is present
• Visible on H&E staining

69. References
• Dacie and Lewis practical hematology
• Bone Marrow Pathology - Barbara Bain - 4th
edition
• Wintrobe's clinical hematology
• Google images

70. Thank you