Ann Lab Med.  2019 Mar;39(2):141-149. 10.3343/alm.2019.39.2.141.

Development of a Novel Flow Cytometry-Based System for White Blood Cell Differential Counts: 10-color LeukoDiff

• Park D
• Chang J
• Kahng J
• Park H
• Jo I
• Kim Y
• Han K

• KMID: 2425969
• DOI: http://doi.org/10.3343/alm.2019.39.2.141

Abstract

BACKGROUND
Flow cytometry (FCM) is commonly used to identify many cell populations. We developed a white blood cell (WBC) differential counting system for detecting abnormal cells using FCM incorporating 10 colors and 11 antibodies in a single tube, called "10-color LeukoDiff," and evaluated its performance.
METHODS
Ninety-one EDTA-anti-coagulated peripheral blood samples from 76 patients were analyzed using 10-color LeukoDiff. We compared 10 color LeukoDiff results with the results of manual differential count (manual diff). WBCs were classified into 17 cell populations: neutrophils, total lymphocytes, T lymphocytes, B lymphocytes, CD5 and CD19 co-expressing lymphocytes, natural killer cells, total monocytes, 16+ monocytes, eosinophils, immature granulocytes, basophils, myeloblasts, B-blasts, T-blasts, myeloid antigen-positive B-blasts, CD19- plasma cells, and 19+ plasma cells.
RESULTS
The correlations between the 10-color LeukoDiff and manual diff results were strong (r>0.9) for mature neutrophils, lymphocytes, eosinophils, immature granulocytes, and blasts and moderate for monocytes and basophils (r=0.86 and 0.74, respectively). There was no discrepancy in blast detection between 10-color LeukoDiff and manual diff results. Furthermore, 10-color LeukoDiff could differentiate the lineage of the blasts and separately count chronic lymphocytic leukemic cells and multiple myeloma cells.
CONCLUSIONS
The 10-color LeukoDiff provided an accurate and comprehensive WBC differential count. The most important ability of 10-color LeukoDiff is to detect blasts accurately. This system is clinically useful, especially for patients with hematologic diseases, such as acute leukemia, chronic lymphocytic leukemia, and multiple myeloma. Application of this system will improve the development of FCM gating strategy designs.

Keyword

Flow cytometry; Manual differential count; 10-color LeukoDiff; Blasts; Immature granulocytes

MeSH Terms

Antibodies
B-Lymphocytes
Basophils
Eosinophils
Flow Cytometry
Granulocyte Precursor Cells
Granulocytes
Hematologic Diseases
Humans
Killer Cells, Natural
Leukemia
Leukemia, Lymphocytic, Chronic, B-Cell
Leukocytes*
Lymphocytes
Monocytes
Multiple Myeloma
Neutrophils
Plasma Cells
T-Lymphocytes
Antibodies

Figure

• 

  Fig. 1 10-color LeukoDiff plots of whole nucleated cell (WN) (A), orientating gate (B), neutrophils (C, D, and E), lymphocytes (T, B, NK, 5+ 19+) (F, G, and H), monocytes (I and J), eosinophils (K), immature granulocytes (L), chronic myelogeneous leukemia (M), basophils (N and O), acute myeloid leukemia (P), blasts (Q and R), myeloblasts (S), B blasts (T), myeloid antigen-positive B blasts (U), and 19-plasma cells (V, W, X, and Y) in peripheral blood samples.Abbreviation: NK, natural killer cells.

• 

  Fig. 2 Binomial scatter plots and correlations between manual differential count and 10-color LeukoDiff results for neutrophils (A), lymphocytes (B), monocytes (C), eosinophils (D), basophils (E), immature granulocyte (F), blasts (G), and plasma cells (H).

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