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Variability in subjective review of umbilical cord blood colony forming unit assay
Author(s) -
Powell K.,
Kwee E.,
Nutter B.,
Herderick E.,
Paul P.,
Thut D.,
Boehm C.,
Muschler G.
Publication year - 2016
Publication title -
cytometry part b: clinical cytometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.646
H-Index - 61
eISSN - 1552-4957
pISSN - 1552-4949
DOI - 10.1002/cyto.b.21376
Subject(s) - umbilical cord , colony forming unit , coefficient of variation , andrology , biology , veterinary medicine , medicine , chemistry , chromatography , anatomy , bacteria , genetics
Background The goal of this study was to quantitatively evaluate the reproducibility of current manual counting methods of colony forming units (CFUs) from umbilical cord blood samples Methods Fresh and reconstituted frozen cells from 10 cord blood samples were cultured under standard conditions. The number of BFU‐Es, CFU‐GMs, and CFU‐GEMMs were counted by three expert reviewers using the standard microscope method and manually traced CFUs on digital images of cell cultures. Results The mean colony count based on the traced digital images was 82 (22% CV) and 52 (15% CV) for the fresh and frozen samples, respectively. This was significantly greater than that observed using the microscope, 61 (13% CV) for fresh and 43 (16% CV) for frozen. The difference was mainly due to the reviewers observing more CFU‐GMs in the digital images than through the microscope review. All three reviewers agreed on the presence of a colony 72% of the time based on the digital review in both fresh and frozen samples. Reviewer agreement with respect to colony type in the fresh samples was 38% (22%CV), 25% (51%CV), and 6% (115%CV) for BFU‐Es, CFU‐GMs, and CFU‐GEMMs, respectively. Reviewer agreement increased for BFU‐Es and CFU‐GMs in the frozen samples where fewer colonies were present. Conclusions Although this study showed marked variability between reviewers, the analysis of manually traced digital images has the potential to improve inter‐observer variation when compared to current methods by identifying features that lead to discrepancies in colony counting and providing cases with consensus results. © 2016 International Clinical Cytometry Society