Korean J Lab Med.  2011 Jul;31(3):131-137. 10.3343/kjlm.2011.31.3.131.

Reliable, Accurate Determination of the Leukocyte Differential of Leukopenic Samples by Using Hematoflow Method

• Jo Y
• Kim SH
• Koh K
• Park J
• Shim YB
• Lim J
• Kim Y
• Park YJ
• Han K

• KMID: 1735842
• DOI: http://doi.org/10.3343/kjlm.2011.31.3.131

Abstract

BACKGROUND
Hematology analyzers may ineffectively recognize abnormal cells, and manual differential counts may be imprecise for leukopenic samples. We evaluated the efficacy of the Hematoflow method for determining the leukocyte differential in leukopenic samples and compared this method with the manual differential method.
METHODS
We selected 249 blood samples from 167 patients with leukopenia (WBC counts, 500-2,000/microL) for analysis in this study. The EDTA-anticoagulated blood samples were analyzed using an automatic blood cell counter (DxH800; Beckman Coulter, USA) and flow cytometry (FC 500; Beckman Coulter) by using Cytodiff reagent and analysis software (Beckman Coulter). Hematoflow results were selected or calculated from DxH800 and Cytodiff results. Two trained pathologists performed a manual differential count by counting 50-100 cells.
RESULTS
The precision of the Hematoflow method was superior to that of the manual method in counting 5 leukocyte subpopulations, immature granulocytes (IGs), and blasts. Blasts were detected in all 45 cases (100%) by Hematoflow. The correlation of the Cytodiff blast count to the reference count was high (r = 0.8325). For all other cell populations, the correlation of the Hematoflow results with the reference count was stronger than that of the other manual counts with the reference count.
CONCLUSIONS
The Hematoflow differential counting method is more reproducible and sensitive than manual counting, and is relatively easy to perform. In particular, this method detected leukemic blasts more sensitively than manual differential counts. The Hematoflow method is a very useful supplement to automated cell counting.

Keyword

Hematoflow; Cytodiff; Leukopenia; Flow cytometry; Hematology cell analyzer

MeSH Terms

Adolescent
Adult
Aged
Child
Child, Preschool
Female
Flow Cytometry/*methods
Granulocytes/cytology
Humans
Infant
Leukocyte Count/*methods
Leukocytes/*cytology
Leukopenia/*blood/diagnosis
Male
Middle Aged
Reagent Kits, Diagnostic
Software

Figure

• 

  Fig. 1 An example of CytoDiff results. Sixteen cell populations are displayed in different colors with complicated gates.

• 

  Fig. 2 (A) Non-B-non-T blasts (red) and basophils (black) using the CD2+CD294 expression pattern. (B) The basophils show higher fluorescence than the non-B-non-T blasts.

• 

  Fig. 3 (A) B lymphoblasts (brown) and B lymphocytes (sky-blue) using the CD19+ cell population are differentiated by CD45 expression level. (B) Because the promyelocytes are included in IGs, acute promyelocytic leukemic cells are counted as IGs (purple).

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  Fig. 4 (A) Paroxysmal nocturnal hemoglobinuria (PNH) type III granulocytes are counted as IGs in samples from PNH patients. (B) A similar proportion of mature neutrophils from the same patient are FLAER-negative (type III, P4).

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