Flow Cytometry Evaluated for Cord Blood Differentials

The potential of flow cytometry to analyze and to report leukocyte differentials on cord blood to be used for hematopoietic stem cells has been evaluated.

Currently, the reference method for white blood cell (WBC) is the microscopic manual count, but international standards advise that potential donor blood is tested for total nucleated cell count, nucleated red blood cell count (nRBCs), total number of CD34 cells, viability or potency as measured by viable CD34 cells. Colony-forming unit assays, microbial cultures, and finally a complete blood count with WBC differential are also advisable.

Hematologists at the Centre Hospitalier Universitaire (CHU; Rennes, France) analyzed 161 cord bloods between November 2010 and February 2011. WBC differentials were determined for each sample, by a cell counter, a manual method, and the flow cytometry using an antibody cocktail. WBC differential includes the count of neutrophils, eosinophils, basophils, lymphocytes, monocytes, immature granulocytes, and blasts cells.

All samples were analyzed on the HematoFlow platform (Beckman Coulter, Miami, FL, USA) within 12 hours of collection. This platform is built around Beckman Coulter's FP1000 cell preparator, along with their FC500 five-color flow cytometer. The antibody cocktail used was another Beckman Coulter product called the CytoDiff.

A good correlation was obtained for neutrophils, lymphocytes, and immature granulocytes when the manual method was compared to the flow cytometry, but was weaker for eosinophils and even lower for monocytes and basophils. The flow cytometry method employed allowed the identification of 12 cell
subtypes (neutrophils, eosinophils, basophils, B lymphocytes, T-non-cytotoxic and T cytotoxic/Natural Killer (NK) lymphocytes, CD16 negative monocytes and CD16 positive monocytes, immature granulocytes, B blasts, T blasts and non-B-non-T blasts) within a single panel of antibodies and a minimal volume of 50 µL of blood.

The authors concluded that the flow differential has some drawbacks such as the cost of CytoDiff reagent, but this can be balanced by the reduction in technician time required for manual counting and the use of a standardized product. The flow cytometry appears as a technique completely adapted to provide a WBC differential on a cord blood sample. The additional cell subset counts offered in the same turn-around-time would probably be exploited in the near future to better characterize the engraftment potential. The study was published in the February 2013 issue of the International Journal of Laboratory Hematology.

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