Implementing a routine flow cytometry assay for nucleated red blood cell counts in cord blood units

Abstract

Flow cytometric NRBC enumerations, now implemented in Héma-Québec Public Cord Blood Bank, is an improvement in the efficiency of our operations by integrating the count for NRBC into our flow cytometry platform.

Automated nucleated red blood cell count using the Mindray BC‐6800 hematology analyzer

“…Flow cytometry has been proposed as a reference for studying the results derived from new automated HAs and for ascertaining the accuracy and reliability of traditional MM . Therefore, a modified FC method was used as a reference to evaluate the NRBC counts provided by the automated analyzer in this study . Using a combination of nuclear staining with 7‐AAD and CD45‐FITC surface labeling, NRBCs were identified as 7‐AAD‐stained cells (nucleated cells) without CD45 cell surface expression (nonleukocyte cells) .…”

“…11,15 Using a combination of nuclear staining with 7-AAD and CD45-FITC surface labeling, NRBCs were identified as 7-AAD-stained cells (nucleated cells) without CD45 cell surface expression (nonleukocyte cells). 15 The absolute NRBC counts obtained from FC were compared with those obtained from the BC-6800 automated analyzer and MM, the most routinely used technique in most laboratories. The results showed excellent agreement between the reference FC method and both the Mindray BC-6800 analyzer and the MM method and did not reveal a statistically significant proportional (slope) or constant The results of the present study reveal that NRBC counts obtained by the Mindray BC-6800 analyzer are excellent and that this device could, therefore, replace traditional manual NRBC counts.…”

“…Therefore, a modified FC method was used as a reference to evaluate the NRBC counts provided by the automated analyzer in this study . Using a combination of nuclear staining with 7‐AAD and CD45‐FITC surface labeling, NRBCs were identified as 7‐AAD‐stained cells (nucleated cells) without CD45 cell surface expression (nonleukocyte cells) . The absolute NRBC counts obtained from FC were compared with those obtained from the BC‐6800 automated analyzer and MM, the most routinely used technique in most laboratories.…”

“…The FC method (the reference method) was modified from two previously reported studies, namely, Tsuji et al and Simard et al Briefly, 25 μL of EDTA blood was incubated with 10 μL of FITC‐labeled anti‐CD45 (CD45‐FITC, clone 2D1, BD Pharminogen, San Jose, CA, USA) for 30 minutes at room temperature in a dark environment. Next, 250 μL of 1X BD Pharm Lyse (BD Biosciences, San Jose, CA, USA) was added and gently mixed.…”

Improving the efficiency of the autoverification workflow for nucleated red blood cell reporting in the hematology laboratory

“…NRBCs were reported as the number of NRBCs per 100 WBCs (%NRBC), including all forms and stages of maturation. Flow cytometric analysis for the detection and quantification of NRBCs was carried out using CD45/CD71/7‐AAD as described in a previous study 22 . A total number of 100 000 nucleated cells were acquired for each sample on BD LSRFortessa ™ with BD FACSDiva software (Beckton Dickinson).…”