Interpretation of atypical patterns encountered when using a flow cytometry-based method to detect residual leukocytes in leukoreduced red blood cell components

Background: Universal leukoreduction of blood components is becoming the standard of care. Flow cytometry methods are being used for quality control of the leukoreduction process. Methods: We provide an atlas of atypical flow cytograms generated by a commercial LeucoCOUNT™ assay that was used to enumerate residual leukocytes in leukoreduced red blood cell components. Numeric results are derived from a flow cytogram generated by the assay. Results: Three types of atypical flow cytogram patterns were observed during process validation or routine quality control of leukoreduced red blood cell components. (a) Fixation artifact: Fixation of control or test samples can alter the staining intensity compared with fresh cells. (b) "Rain" pattern: Flow cytometry methods count slightly damaged leukocytes not removed during leukoreduction. Slightly damaged leukocytes appear on a flow cytogram like "rain" falling from a well-defined "cloud" of intact residual leukocytes. Discrepancies between automated flow cytometry results and subjective manual counting methods can occur. (c) Autofluorescence-debris pattern: Cell debris and age-related changes in the sample can cause shifts in the fluorescence staining pattern, resulting in erroneous test results. Conclusion: Review of flow cytograms is essential for accurate reporting of flow cytometry-based methods for enumerating residual leukocytes in leukoreduced blood components.