Evaluation of the leukocyte differential on a new

automated flow cytometry hematology analyzerJ.-E. KIM, B.-R. KIM, K.-S. WOO, J.-Y. HAN

INTRODUCTION

Differential white blood cell count is a gold standard

for the accurate identification of cells in the peripheral

blood [1]. However, this method is both laborious and

time-consuming. Therefore, complete blood counts

(CBCs) and white blood cell (WBC) differentials con-

ducted using automated hematology analyzers have

replaced the traditional manual differential count

method for the initial screening of hematologic abnor-

malities in clinical laboratories [2, 3]. However,

a majority of automated hematology analyzers have

a limitation in identifying WBCs to only five cell

populations: neutrophils, lymphocytes, monocytes, eo-

sinophils, and basophils.

The Hematoflow (Beckman Coulter, Miami, FL, USA)

is the newest automatic hematology analyzer including

a DxH 800 Coulter Cellular Analysis System and FC 500

Flow Cytometer from Beckman Coulter. The DxH800

uses multiple angles of light scatter, and the FC500

provides a 16-part WBC differential, giving precise

information on WBC subsets [4]. We evaluated the diff-

erential WBC count performance of the FC500 in com-

parison with the Beckman Coulter DxH800 (Beckman

Coulter), Sysmex XE-2100 (TOA Medical Electronics

Co, Kobe, Japan), and the reference manual method.

Departments of Laboratory

Medicine, College of Medicine,

Dong-A University, Busan, Korea

Correspondence:

Jin-Yeong Han, Department of

Laboratory Medicine, Dong-A

University College of Medicine, 1,

3-Ga, Dongdaesin-dong, Seo-gu,

Busan, 602-715, Korea.

Tel.: +82 51 240 5323;

Fax: +82 51 255 9366;

E-mail: jyhan@dau.ac.kr

doi:10.1111/j.1751-553X.2012.01432.x

Received 14 December 2011;

accepted for publication 10 April

2012

Keywords

Blood cell analyzer, leukocyte

differential, blast, cell lineage

SUMMARY

Introduction: The Hematoflow (Beckman Coulter, USA) is a new

automated hematology analyzer, which provides a 16-part white

blood cell count (WBC) differential.

Methods: We evaluated the differential WBC count performance of

the Hematoflow. 101 blood samples from patients were selected for

comparison analysis.

Results: The methodological comparison of the WBC differential para-

meters of neutrophils, lymphocytes, monocytes and eosinophils

showed good correlations among 4 different analyses. More than 1%

of blast cells were counted in 30 of 101 samples. A good correlation

for blast cell counts obtained by Hematoflow was found with the

reference manual method (r=0.9637, P 0.0001). For blast B, Hemato-

flow shows good correlation with reference method results but did

not identify blast T.

Conclusions: These results demonstrate that the Hematoflow has a

comparable performance with the Sysmex XE-2100 and indicate that

B cell lineage ALL can be identified by the use of the Hematoflow in

an initial evaluation of acute leukemia.

SHORT REPORT INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

� 2012 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 1

International Journal of Laboratory HematologyThe Official journal of the International Society for Laboratory Hematology

MATERIALS AND METHODS

One hundred one blood samples (54 men and 47

women; age, 1–84 years; mean age, 43 years) in rou-

tine CBC were selected for analysis regardless of previ-

ous diagnosis. All samples were collected in evacuated

3-mL tubes containing EDTA K3 and were analyzed

within 6 h after phlebotomy. The diagnosis and classi-

fication of the patients were made on the basis of the

2008 WHO classification [5] by morphologic, immu-

nophenotypic, cytogenetic, and molecular genetic

findings of the bone marrow. This study was approved

by the Institutional Review Board of the Dong-a

University of College of Medicine.

The FC500 contains a premixed Cytodiff reagent

and analysis software. The Cytodiff panel included six

directly conjugated monoclonal antibodies in a five-

color single reagent. The leukocytes were differenti-

ated into 16 cell populations (B-lymphocytes, CD16)

T-lymphocytes, CD16+ T and NK cells, T and NK lym-

phocytes, total lymphocytes, CD16 monocytes, CD16+

monocytes, total monocytes, immature granulocytes

[IGs], total eosinophils, mature neutrophils, total neu-

trophils, blasts B, blasts T, blasts non-B-non-T, and

total basophils). Analysis procedures were conducted

in accordance with the manufacturer’s manual [6].

The automated WBC differential counts were com-

pared with the manual differential counts. For the

validation of the results from both the analyzers, two

experienced hematopathologist did manual differential

counts by counting 100 cells. The five-part automated

WBC differential was conducted using both the DxH

800 and the XE-2100 within 6 h after blood collec-

tion. The blast suspect flags generated by the DxH 800

apparatus were compared with the results of the man-

ual differential count. The correlation of the blast cells

was made by the number of cells counted by different

methods.

Statistical analysis

Statistical analysis was performed using MEDCALC v11.2

(Mariakerke, Belgium). The results from the different

methods for WBC differential count are compared by

regression analysis. Values of P < 0.05 were regarded

as significant.

RESULTS

The DxH 800 reported CBC results with the five-part

WBC differential count in 96.0% (97 of 101) of the

cases. The XE-2100 provided a valid automated differ-

ential count in 84.2% (85 of 101) of the cases. The

methodological comparison of the WBC differential

parameters of neutrophils, lymphocytes, monocytes,

and eosinophils showed good correlations between

four different analyses (Table 1). The results obtained

by the XE-2100 were better than DxH800 when

Table 1. Comparison of the results from FC500, DxH800, and XE-2100 with manual differential leukocyte count

Neutrophil Lymphocyte Monocyte Eosinophil Basophil

FC 500

Intercept 3.0203 0.1265 2.0742 0.4041 0.3208

Slope 0.9579 0.9907 0.8151 0.9170 0.4989

r 0.9390 0.9666 0.9079 0.9484 0.5028

P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

DxH 800

Intercept 7.4753 5.7954 5.5057 0.3319 0.9987

Slope 0.8184 0.8993 0.8282 0.9393 1.3110

r 0.8073 0.8084 0.5868 0.9450 0.2684

P <0.0001 <0.0001 <0.0001 <0.0001 0.0093

XE-2100

Intercept 3.1381 6.1907 4.5416 0.1460 0.8843

Slope 0.9492 0.9112 0.7496 0.9566 0.5297

r 0.9574 0.9030 0.6838 0.9739 0.2007

P <0.0001 <0.0001 <0.0001 <0.0001 0.0537

� 2012 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.

2 J.-E. KIM ET AL. A NEW HEMATOLOGY ANALYZER BY FLOW CYTOMETRY

compared with manual counting and closer to the FC

500, with the exception only of counting basophils.

For basophil count, the correlation was moderate

(r = 0.5028) with the FC500; however, the Sysmex

XE-2100 and DxH800 showed poor correlations

(r = 0.2007 and 0.2684, respectively).

In manual method, more than 1% of blast cells

were counted in 30 of 101 samples. There were seven

cases of acute lymphocytic leukemia (ALL), including

four T-cell ALL (T-ALL) cases and three B-cell ALL

(B-ALL) cases; 20 cases of AML; and three cases of

myelodysplastic syndrome (MDS). A good correlation

for blasts was found with the reference manual

method (r = 0.9637, P < 0.0001). There were five

cases of ALL showing over 10% of blast counts

(Table 2). For B-cell ALL, blast B count of Hemato-

flow shows good correlation with reference method

results. For T-cell ALL, Hematoflow did not identify

blast T and shows complete agreement with manual

counting. For AML and MDS, blast non-B-non-T

counts of Hematoflow shows 100% concordance and

good correlation with reference method results.

DISCUSSION

Modern characterization of acute malignant hemato-

logical disease is a multidisciplinary process. Despite

the advances in diagnostic technologies, the mainte-

nance and improvement of morphological skills still

remain essential requirements in the diagnosis of

acute leukemia. Automated differential white blood

cell count can not count certain cell populations pass-

ing through the blood (immature granulocytes, blasts,

atypical lymphocytes, and lymphoma cells), which

must be analyzed secondarily by optical or digitized

microscopy. In hospitals, these reviews represent 10–

25% of cases according to established criteria [7]. In

this context, Beckman Coulter proposed the new He-

matoFlow concept using flow cytometry of 20 000

cells stained by the CytoDiff monoclonal antibodies

combination prior to microscopy. In the new concept,

the alarms generated by the hematology analyzers

induce the review by flow cytometry before any

check by microscopy.

The DxH 800 reported CBC results with the five-

part WBC differential count in majority (96.0%) of

the cases, whereas the XE-2100 provided a valid auto-

mated differential count in 84.2% of the cases. The

correlation of the FC 500 method was better to that of

the XE-2100 in the counting of lymphocytes, mono-

cytes, and basophils. This means the leukocyte differ-

ential obtained using the Hematoflow method is more

reproducible and sensitive than that obtained using

other methods. It represents a substantial advance in

hematology instrumentation and has considerable

potential for extending the clinical role of hematologic

studies.

We found a moderate correlation (r = 0.5028)

between the FC500 basophil count and reference

counts. Some authors reported a low correlation

between the Cytodiff basophil count and reference

counts [8, 9], whereas some found a high correlation

between these counts (r = 0.8727) [6]. This is owing

to CD2+ CRTH2 expressed in activated T cells, eosin-

ophils, and basophils. Separation between basophils

and non-B-non-T blast populations was not effective

in the Hematoflow method [6].

Whatever the pathology (AML, ALL, and MDS)

and the number of blasts on smear, all patients were

positive for blast detection on the Hematoflow.

Table 2. The comparison between blast counts by manual method and the percentage of each blast subset by

Hematoflow in five cases of ALL

No. of cases Diagnosis Manual (%) Blast B (%) Blast T (%) Blast non-B-non-T (%)

1 T-ALL 57 0.69 0.04 58.54

2 T-ALL 28 2.05 0 28.79

3 B-ALL 84 77.87 0 0.63

4 T-ALL 98 0.12 0 94.25

5 B-ALL 47 51.26 0.01 0.33

ALL, Acute lymphocytic leukemia.

� 2012 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.

J.-E. KIM ET AL. A NEW HEMATOLOGY ANALYZER BY FLOW CYTOMETRY 3

Additionally, it appears easy to distinguish B-cell line-

age leukemic blasts from T-cell leukemic blasts and

myeloblasts. The Hematoflow was not able to classify

correctly T-cell leukemic blasts as blast T. The recom-

mended procedure in this case is still to use morpho-

logy as the most reliable result. In cases of T-ALL and

AML, it is needed to be reclassified by the hematologic

pathologists and immunotyping using bone marrow

samples is still required for the initial diagnosis. The

detection rate of blast and the correlation of the blast

count by the Hematoflow method were similar to the

previous study [6]. It may be useful to screen samples

for the presence of blasts using Cytodiff. If blasts are

detected by Cytodiff, confirmation of the blasts by slide

review is recommended, especially in new patients.

While acknowledging the limitations of our study,

our data indicate that B-cell lineage ALL can be iden-

tified by the use of the Hematoflow in an initial eval-

uation of acute leukemia. For more accurate

distinctions of lineages, further studies with more

specimens will be necessary to apply for all occasions.

In this study, we became aware of the capabilities

and limitations of the automated Hematoflow for ana-

lyzing patient sample with leukemic blasts. For the

technical staff, the installation of Hematoflow would

have many consequences: the reduction in technical

staff time at the microscope while simultaneously

increasing the efficiency of the workflow, the elimina-

tion of medical technologists facing a difficult diagno-

sis alone. This rapid and quantitative technique may

be helpful in the differential diagnosis of acute leuke-

mias prior to additional investigation.

ACKNOWLEDGEMENTS

This study was supported by the Korea Science and

Engineering Foundation (KOSEF) grant funded by the

Korea government (MEST; R13-2002-044-05002-0).

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