Flow cytometric CD34+ stem cell enumeration: lessons from nine years' external quality assessment within the Benelux countries.
BACKGROUND
A biannual external quality assurance (EQA) scheme for flow cytometric CD34+ haematopoietic stem cell enumeration has been operational in the Benelux countries since 1996. In an evaluation of the results of 16 send-outs, we studied the effects of the methods used on assay outcome and whether or not this exercise was effective in reducing between-laboratory variation.


METHODS
Data were analyzed using robust multivariate regression. This approach is relatively insensitive to outliers and is used to assess the effect of methodological aspects of CD34+ cell counting on the bias and variability.


RESULTS
Five variables were associated with significant bias of absolute CD34+ cell counts: (i) unique laboratory number (ULN), (ii) gating strategy; (iii) CD34 mAb fluorochrome; (iv) type of flow cytometer, and (v) method of sample preparation. In addition, ULN and platform methodology (i.e., single vs. dual) contributed significantly to the variability of this assay. Overall, the variability in results of CD34+ cell enumeration has declined with time; in particular, after a practical workshop in which participants were trained to use the "single platform ISHAGE protocol."


CONCLUSIONS
Between-laboratory variation in CD34+ cell enumeration can be reduced by standardization of methodologies between centres. Our approach, i.e., EQA with targeted training and feedback in response to reported results, has been successful in reducing the variability of CD34+ cell enumeration between participants.
Levering
Wilfried H B M
WH
Laboratory for Histocompatibility and Immunogenetics, Sanquin Blood Bank South West Region, Rotterdam, The Netherlands. w.levering@sanquin.nl
Preijers
Frank W M B
FW
van Wieringen
Wessel N
WN
Kraan
Jaco
J
van Beers
Wil A M
WA
Sintnicolaas
Kees
K
van Rhenen
Dick J
DJ
Gratama
Jan W
JW
D018952:Antigens, CD34 / Q000032:analysis*; D001530:Belgium; D001772:Blood Cell Count / Q000295:instrumentation / Q000379:methods / Q000592:standards*; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000592:standards; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D008189:Luxembourg; D009426:Netherlands; D011786:Quality Control; D011933:Reagent Kits, Diagnostic / Q000592:standards; D012016:Reference Values; D012189:Retrospective Studies; D013997:Time Factors
D018952:Antigens, CD34; D005456:Fluorescent Dyes; D011933:Reagent Kits, Diagnostic