An internal positive control for the enumeration of CD45(+) and CD34(+) cells by flow cytometry allows monitoring of reagent and operator performance.
BACKGROUND
Three-color flow cytometry assays are used to determine CD34(+) cell doses prior to stem cell transplantation. These assays require high-quality reagents that are dispensed accurately to ensure reproducible results. We have developed a flow cytometry assay for CD34(+) cells with an integral positive control (KG1a cells) for monitoring reagent and operator performance.


METHODS
The method was validated using samples from 127 allogeneic donations (42 BM, 85 PBSC) from healthy donors and 195 autologous donations (46 BM, 149 PBSC) from patients in remission from hematologic malignancies. The mean, SD and range of CD45(+) and CD34(+)cell counts were determined for each donation type. An internal control was used to assess performance of reagents and operators by comparison with a predetermined target value and an experienced operator.


RESULTS
Replicate studies showed the method to be accurate and precise, with KG1a cells at 97.7+/-3.9% of the target value and a CV of 4.0%. In routine use over 322 samples, the accuracy was 91.7+/-17.7% of the target value, with a CV of 19.3%. Investigations into the cause of the reduced precision showed that reagents performed consistently well but operator performance was variable, with two of six operators significantly under-dispensing KG1a cells.


CONCLUSIONS
This study validates our three-color flow cytometry assay and demonstrates that KG1a cells may be used to monitor test performance in the routine working environment. In addition to monitoring performance within a single laboratory, its wider use in multicenter studies may be helpful regarding standardization of methods.
Guttridge
M G
MG
Stem Cells and Immunotherapy, National Blood Service, NHS Blood and Transplant, Bristol, BS10 5ND, UK. martin.guttridge@nbs.nhs.uk
Belfield
H
H
Hollyman
D
D
Lankester
A
A
Watt
S M
SM
D018952:Antigens, CD34 / Q000097:blood*; D005434:Flow Cytometry / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D017493:Leukocyte Common Antigens / Q000097:blood*; D011361:Professional Competence; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results
D018952:Antigens, CD34; D017493:Leukocyte Common Antigens