11. Disadvantages
• Arrangement of cells in the marrow
• Relationship between the cells
• Fibrotic marrows: aspiration of blood
12.
13.
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
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
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
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
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
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
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
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
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
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
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
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