Evaluation of droplet digital PCR for quantification of residual leucocytes in red blood cell concentrates

• Published in: Vox sanguinis 2017-11, Vol.112 (8), p.744-750
• Main Authors: Doescher, A., Loges, U., Petershofen, E. K., Müller, T. H.
• Format: Article
• Language: English
• Subjects: Automation; Blood; Blood groups; Blood Safety; Cytometry; Deoxyribonucleic acid; digital droplet PCR; Dilution; DNA; DNA - blood; DNA - genetics; Enumeration; Erythrocyte Transfusion; Erythrocytes; Erythrocytes - cytology; Flow Cytometry; Humans; leucocyte counting; leucodepleted red blood cell concentrates; Leukocyte Count - methods; Leukocytes; Pilot Projects; Polymerase Chain Reaction; Quality Control; quantitative PCR; Reproducibility; Reproducibility of Results; Sensitivity; Sensitivity and Specificity; Transfusion Reaction - prevention & control
• ISSN: 0042-9007; 1423-0410
• DOI: 10.1111/vox.12566

Background and Objectives Enumeration of residual white blood cells in leucoreduced blood components is essential part of quality control. Digital PCR has substantially facilitated quantitative PCR and was thus evaluated for measurements of leucocytes. Materials and Methods Target for quantification of leucocytes by digital droplet PCR was the blood group gene RHCE. The SPEF1 gene was added as internal control for the entire assay starting with automated DNA extraction. The sensitivity of the method was determined by serial dilutions of standard samples. Quality control samples were analysed within 24 h, 7 days and 6 months after collection. Routine samples from leucodepleted red blood cell concentrates (n = 150) were evaluated in parallel by flow‐cytometry (LeucoCount) and by digital PCR. Results Digital PCR reliably detected at least 0·4 leucocytes per assay. The mean difference between PCR and flow‐cytometric results from 150 units was −0·01 (±1·0). DNA samples were stable for up to at least six months. PCR measurement of leucocytes in samples from plasma and platelet concentrates also provided valid results in a pilot study. Conclusion Droplet digital PCR to enumerate leucocytes offers an alternative for quality control of leucoreduced blood products. Sensitivity, specificity and reproducibility are comparable to flow‐cytometry. The option to collect samples over an extended period of time and the automatization introduce attractive features for routine quality control.