A Bone of Contention: Best Approach for Harvesting Bone Marrow to Maximize TNC and CD34 Cell Counts

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A Bone of Contention Best Approach for
Harvesting Bone Marrow to Maximize TNC and CD34
Cell Counts
V Antonenas1, F Garvin1, K Mickelthwaite1, I
Kerridge1,2,3, K F Bradstock1,2, DJ Gottlieb1,2,3
1Sydney Cellular Therapies Laboratory, 2Blood
and Marrow Transplant Service, Westmead Hospital,
Sydney, NSW, Australia, 3University of Sydney,
NSW, Australia
Westmead Hospital
The BMT Laboratory, Westmead Hospital
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Introduction

• Bone marrow (BM) has been utilized as a source
  of stem cells for transplantation for many years.
  Although the use of BM has decreased with the
  advent of mobilized stem cells, utilization is
  increasing once again due to the lower rate of
  chronic GVHD associated with BM as a stem cell
  source.
• There is no generally accepted technique for
  harvesting BM. Protocols vary both in relation to
  the volume of each aspirate and the number of
  aspirates performed at each puncture site.

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Aim

• To investigate whether differences in the volume
  and number of aspirates taken at the time of BM
  harvest affect the number of mononuclear cells
  (particularly CD34 cells and CD3 cells)
  collected.

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Method

• Consented adult donors were positioned in the
  prone position for collection of bone marrow from
  the posterior iliac crests.
• Triplicate samples of 5, 10 and 20ml aspirate
  volumes were taken at the start.
• Subsequent harvesting was performed using 10ml
  aspirate volumes.
• Triplicate samples were taken from the 10ml
  aspirates when harvest volumes of 250, 500, 750
  and 1000mls were attained.
• Samples were analysed for total nucleated cell
  (TNC) count, CD34 and CD3 cell counts.
• See Figure 1

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METHOD
Remainder into harvest bag
5,10 or 20ml aspirates
1 to 1.5ml samples
Iliac crest
Lab processing
Cortical layer
Analyse for TNC, CD34 CD3
Bone marrow
Figure 1. BM is aspirated from the posterior
iliac crest, just beneath the cortical layer. The
needle is advanced one centimetre at a time for
subsequent aspirates.
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Results
Table 1. Results from 7 donors Total cell counts
(SEM) taken from 5, 10 and 20 ml aspirate
volumes at the beginning of harvest are shown in
the third column. Cell counts from 10ml aspirates
after 250, 500, 750 1000ml of BM have been
taken are shown in the last 4 columns.
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CD34 and CD3 Analysis
a
b
c
d
CD34
R9
f
g
e
CD3
CD34 R6 x 100 R1
CD3 R7 x 100 R1
Figure 2. In-house gating strategy for CD34 and
CD3 analysis of BM harvest. Histogram (a) shows
all CD45 leukocytes region 1 (R1). Histogram (b)
shows CD34 events (R3) gated on R1. Histograms
(c) and (d) are sequentially gated from R3 with
CD34 events identified in R6. Histogram (e)
shows CD3 events (R2) gated on R1. Histograms
(f) and (g) are sequentially gated from R2 with
CD3 events identified in R7.
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Mean Cell Numbers from Various Aspirate Volumes
N7
TNC x 106
CD34 x 104
CD3 x 105
Figure 3. Cell numbers from 5, 10 or 20 ml marrow
aspirate at commencement of harvest.
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Figure 4. Cell numbers obtained from 10ml marrow
aspirates at various times during marrow harvest.
Mean Cell Numbers at Various Harvest Volumes
N7
TNC x 106
CD34 x 104
CD3 x 105
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Relative Cell Numbers per ml
N7
N7
Figure 9 Comparison of cell numbers (per ml) at
various aspirate volumes
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Conclusion

• The aspiration of increasing volumes of BM does
  not produce a proportional increase in total
  numbers of TNC, CD34 or CD3 cells collected.
• The highest ratio of CD34 to CD3 cells is
  observed with 10ml aspirates.
• There is a progressive drop in yield of both
  CD34 and CD3 cells once 750ml of BM has been
  aspirated in 10 ml aliquots.
• Continued harvesting after collection of 1000ml
  will produce small numbers of CD34 cells. If
  insufficient stem cells have been obtained at
  this time, a second harvest may be a better
  option than continued aspiration on the same
  occasion.