P173 The ART(EFACT) of multicolor flow cytometry. A case of mysterious CD3/CD19 double positive lymphocytes in the flow cytometry crossmatch (FCXM) assay

Abstract

Aim Over the past 3 months we have been noticing an unusual population of CD3/CD19 double positive (DP) lymphocytes in some flowcytometry crossmatches (FCXM) performed in our HLA lab. Initially, the appearance of these unusual DP cells seemed random. However, upon careful review it was noted that the DP cells were seen only in reactions with the positive control sera and only when two recently trained techs were involved in setting up FCXM. Who was to blame? Methods FCXM were performed in 96-well trays according to the Halifax protocol. In house negative control (NC) and positive control (PC) sera were used in each FCXM. T/B cells were identified using mouse anti-CD3 PerCP(BD) and anti-CD19-PE (BD) mAbs respectively. Positive FCXM reactions were identified using polyclonal goat anti-human IgG-FITC (Jackson). FCXM were acquired on BD Canto II and analysed using Diva software. Results Fig. 1A illustrates a representative dot plot showing the unusual CD3/CD19 DPcell population observed with the PC sera in FCXM set up by one of the two recently trained technologists. This DP population appeared to be a result of CD3 + T cells shifting up on the PE (FL2) scale and is not seen with the NC sera (Fig. 1B) or any patient sera in the same FCXM. Repeat FCXM performed with identical reagents (including the PC) by a senior technologist did not reveal the DP cells. While direct observation did not identify any protocol deviations by the two junior technologists, it was noted that both individuals used red sharpies to mark the rims of the PC wells (blue for NC) in crossmatch trays (Fig. 1C). Further experiments showed that marking tray rims with red sharpies caused the appearance of the DP cells. Similar effect was seen with pink or purple sharpies but not when black, blue or green sharpies were used (Fig. 1D). This was attributed to the presence of Rhodamine B (fluorescence emission spectrum similar to PE) in the ink of red, pink and purple sharpies. Conclusions This report HIGHLIGHTS that even the best intentions can create strange and unexpected artefacts in FCXM. Download : Download high-res image (303KB) Download : Download full-size image