re CD34 enumeration on bone marrow

rob.sutherland@utoronto.ca rob.sutherland at utoronto.ca
Thu Nov 10 00:54:17 EST 2005


Hello Alireza,
there are indeed standardized protocols for enumerating CD34+ cells, 
but before I point you in the appropriate direction I need to make sure 
I understand your question correctly. Are you referring to the 
enumeration of CD34+ cells in bone marrow samples to be used in the 
transplant setting, or are you referring to the enumeration of CD34+ 
cells in cytokine-mobilized peripheral blood stem cell (PBSC) 
collections to be used in this setting?
While the methods at the heart of the standardized protocols were 
originally developed to count CD34+ cells in marrow samples in a 
research lab environment (Experimental Hematology  22:1003-1010, 2004) 
their clinical utility in the hematopoietic stem cell (HSC) transplant 
setting is of most value in the enumeration of CD34+ cells in 
peripheral blood and PBSC (apheresis) products. Assessments of Cord 
Blood as well as other samples are also possible with the same 
protocols.
These days, I think it is safe to say that very few HSC transplants are 
performed using bone marrow as a source of HSCs. Although I believe 
that the AABB insist that CD34 enumeration be performed on marrow 
samples used for transplant, I believe this requirement is misguided.  
Since the late 1950s until the time PBSC transplants became the 
preferred source of HSCs (mid 1990s?), bone marrow transplants were 
performed based solely on 'weighing the bag' (of marrow collected) and 
performing a white blood cell count with a hematology analyzer (a total 
nucleated cell count of 2.5 x 10e8 nucleated cells/Kg patient weight 
usually ensured hematopoietic reconstitution).
Even though the standardized protocols referenced below can perform 
CD34+ cell enumeration on marrow samples, the total CD34 cell number 
does not tell us anything about the 'graft adequacy' of a bone marrow 
sample collected for transplant. This is mainly because the qualitative 
composition of BM CD34+ cells is very different from that of PBSC 
collections. BM CD34+ cells are very much more heterogeneous than PBSC 
CD34+ cells and there are a lot more primitive, candidate stem cells as 
a proportion of the total CD34+ cells in PBSC than there are in 
'normal' marrow CD34+ cells. There are a number of other good reasons, 
but essentially there is no 'magic target number' of CD34+ cells in 
bone marrow that will ensure rapid and sustained engraftment.
The standardised protocols most widely used today were based on the Exp 
Hematol paper referred to above that evolved into the original 'ISHAGE 
Guidelines' (Journal of Hematotherapy 3:213-226, 1996). The 'single 
platform' development of this methodology that included viability 
assessment was published thereafter (Cytometry [Communications in 
Clinical Cytometry] 34:61-67, 1998). The essential components of the 
latter are embodied in the 'Basic Protocol' devised for 'Current 
Protocols in Cytometry' (Unit 6.4 1999 and in updated form 2001, pp 
1-23).	They are also embodied in the recommendations from the European 
Working Group on Clinical Cell Analysis (EWGCCA) (Cytometry 
[Communications in Clinical Cytometry] 34:128-142, 1998), the British 
Protocol for CD34+ cell enumeration (Clin. Lab. Haematol. 21:301-308, 
1999), and the German Reference Protocol (Transfusion. 39:1220-1226, 
1999) among others.
Like earlier versions of the ISHAGE protocols, the single platform 
derivatives have been developed to work on both Beckman/Coulter and BDB 
flow cytometers.  However, there are minor technical differences in the 
way the assay is set up on the different instruments (detailed in CPC 
Unit 6.4, 2003). Reagents to enable the enumeration of viable CD34+ 
cells using single platform ISHAGE-style methodology are available from 
Beckman-Coulter ("StemKit"), Dako-Cytomation, and Becton Dickinson 
Biosystems.
I hope this helps (and is not too detailed).

Rob Sutherland
University Health Network
Toronto
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