[Cytometry] CD34 pos. stem cell enumeration question

D. Robert Sutherland rob.sutherland at utoronto.ca
Fri Jul 31 16:58:58 EDT 2009

there have been a couple of studies that have attempted to monitor  
the recovery of viable CD34+ cells post thaw and correlate it with  
engraftment. We did one (Allan et al Number of viable CD34+ cells re- 
infused predicts engraftment in autologous hematopoietic stem cell  
transplantation. Bone Marrow Transplantation 29: 967-972, 2002), and  
another recent one is from Lee S, Kim S, Kim H, Baek EJ, Jin H, Kim  
J, Kim HO. Post-thaw viable CD34(+) cell count is a valuable  
predictor of haematopoietic stem cell engraftment in autologous  
peripheral blood stem cell transplantation. Vox Sang. 2008 Feb;94(2): 
146-52. Epub 2007 Nov 19). There  may be one or two others.

Most labs do not routinely perform viable CD34+ cells enumeration on  
post thawed samples because the pre freeze numbers correlate very  
well with engraftment. Current thinking is that if you infuse 2 x  
10e6 viable CD34+ cells pre-freeze/Kg patient weight you will engraft  
in about 12-14 days. The minimum number required post-thaw is more  
difficult to determine because of the non-standardised manner in  
which post-thawed analysis is performed at different institutions.
Additionally, it takes a minimum of about 30 minutes from sample thaw  
to stain, process and perform flow analysis during which time the  
viability of the CD34+ cells may continue to deteriorate. The use of  
lysing agents on post-thawed samples in some studies may also affect  
the viability of the CD34+ cells reported. Meanwhile, the patient  
will likely have been infused with the post thawed sample pretty well  
immediately post thaw.
Bottom line is there is no good data on how closely the post thaw  
analysis represents the number of viable CD34+ cells infused.
Currently there is no minimum level of post thaw viability that is  
acceptable, but we would recommend alerting the physician to the  
possibility of slow engraftment with respect to any sample containing  
less than 50% viable CD34+ cells.
At the TGH Institution, we only perform the test on samples suspected  
of having been 'mishandled' in some way, or subject to storage in  
faulty freezers etc. We would recommend post thaw flow analysis for  
cord blood samples (alongside CFU assays) identified for potential  
for transplantation.

Rob Sutherland and Mike Keeney
Toronto General Hospital/London Health Sciences Centre

On 24-Jul-09, at 1:17 PM, George, Shadya S wrote:

> I am a medical technologist working at a small stem cell transplant  
> lab.
> I found your response on this subject on the internet and wanted to  
> ask
> you a further question.
> We are currently switching to a using the Stem Kit single platform  
> with
> 7-AAD to enumerate post thaw CD34 absolute counts for autologous
> transplant.  We currently will use this as a quality indicator.  Do  
> you
> have any recommendations as to what an acceptable post freeze  
> viability
> or % Stem Cell recovery should be?  If the recovery is low (<40%),
> should we consider recalculating our stem cell dose/Kg?  Are you aware
> of any recommendations for a cut-off dose calculated post freeze?  For
> example, would 1.0 x 106 be a new acceptable limit when measured post
> freeze?
> I appreciate any thoughts you have on the subject based on your
> experience.
> -Shadya George, MT(ASCP)
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