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Original Article · Originalarbeit
Infus Ther Transfus Med 2001;28:277–279
Received: June 1, 2001
Accepted: August 6, 2001
Automated Analysis of Bone Marrow Fluid with the
Sysmex XE-2100 Blood Cell Counter
G. Zini
G. Mistretta
G. Giordano
L. Laurenti
G. d’Onofrio
Key Words
Nucleated red blood cells · Bone marrow aspiration ·
Immature granulocytes · Erythroblasts · Automated blood
cell counters
Schlüsselwörter
Kernhaltige rote Blutzellen · Knochenmarkpunktat ·
Unreife Granulozyten· Erythroblasten · Automatische
Blutzellzählgeräte
Summary
Background: Until recently, automated analysis of the cellular composition of bone marrow has not been possible,
owing to problems in erythroblast identification, contamination by fat particles, and the heterogeneity of cell types.
The XE-2100 automated blood cell counter can effectively
count erythroblasts and granulocytes using laser light flow
cytometry and DNA fluorochromes. In this study we have
evaluated the performance of the XE-2100 for analyzing
bone marrow cells. Material and Methods: The XE-2100
hematology analyzer performs white cell differential counts
by measuring radiofrequency, direct current resistance, forward and side scatter of laser light, and fluorescence. Erythroblasts are identified by means of their specific fluorescent labelling. We have assessed i) the imprecision of the
XE-2100 nucleated red blood cell (NRBC) counting using linear regression and the National Committee for Clinical Laboratory Standards (NCCLS) document H20A method of differences between duplicates, and ii) the comparability of
NRBC count with microscopy counts using linear regression
and analysis of differences. Results: Reproducibility of the
XE-2100 measurements was excellent with coefficients of
variation below 15%. We compared bone marrow granulocyte and erythroblast proportions by the XE-2100 with microscopy counts using linear regression and analysis of differences on 207 samples: Correlation between XE-2100 and
the microscope was excellent for granulocyte percentage
but slightly lower for erythroblasts. There was a tendency
for the XE-2100 to slightly underestimate erythroblast percentage in most samples with a mean difference (XE-2100 –
microscope) of –13.4%.
Zusammenfassung
Hintergrund: Noch bis vor kurzem war die automatisierte
Analyse der Zellbestandteile von Knochenmarkpunktaten
nicht möglich. Gründe waren die fehlende Erkennung und
Separation von Erythroblasten, die Kontamination durch
Fettpartikel und die Vielfalt der Zellbestandteile selbst. Mit
dem automatisierten Blutzellanalysator XE-2100 ist es jetzt
möglich, Erythroblasten und Granulozyten durch die Anwendung der Durchflusszytometrie mit Laserlicht und DNAFluoreszenzfarbstoffen sicher zu trennen. In dieser Arbeit
haben wir die Verwendbarkeit des XE-2100 zur Knochenmarkanalyse getestet. Material und Methoden: Der Hämatologieanalysator XE-2100 benutzt für die Leukozytendifferenzierung fünf verschiedene Messprinzipien: Hochfrequenzund Gleichstrom-Widerstandsmessprinzip, Vorwärts- und
Seitwärtsstreulicht eines Lasers und Fluoreszenzlicht. Die
Erythroblasten werden durch ihr spezielles Fluoreszenzverhalten identifiziert. Wir haben zwei Versuchsreihen durchgeführt: 1) Die Impräzision der NRBC(nucleated red blood cell)Messung mittels linearer Regression und des NCCLS-H20AVerfahrens der Differenzbildung aus Doppelmessungen und
2) die Vergleichbarkeit der NRBC-Messung mit der mikroskopischen Bestimmung ebenfalls mittels linearer Regression und Differenzanalyse. Ergebnisse: Die Reproduzierbarkeit der XE-2100-Messungen war bei einem Variationskoeffizient < 15% ausgezeichnet. Wir haben dann die Granulozyten- und Erythroblastenergebnisse des XE-2100 mit den
Ergebnissen der mikroskopischen Auswertung an 207 Proben mit Hilfe der linearen Regression und Differenzanalyse
verglichen: Die Übereinstimmung zwischen dem XE-2100
und dem Mikroskop war bei den Granulozyten ausgezeichnet und bei den Erythroblasten etwas weniger gut. Die
NRBC-Messergebnisse des XE-2100 lagen bei der Mehrzahl
der Proben geringfügig unter denen der mikroskopischen
Bestimmung. Die Abweichung betrug im Mittel –13,4%.
© 2001 S. Karger GmbH, Freiburg
Fax +49 761 4 52 07 14
E-mail Information@Karger.de
www.karger.com
Accessible online at:
www.karger.com/journals/iut
G Zini
Servizio di Ematologia
Università Cattolica del Sacro Cuore
Largo F-Vito 1, I-00168 Roma, Italy
E-mail recamh@rm.unicatt.it
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Research Center for the Development and Clinical Evaluation of Automated Methods in Hematology (ReCAMH) – Hematology Service –
Università Cattolica del Sacro Cuore, Roma
Introduction
100
XE-2100
80
60
40
y = 0.8945x + 7.9065
2
r = 0.6566
20
0
0
a
20
40
60
80
100
Microscope
80
y = 0.6074x - 1.8603
2
r = 0.4441
60
XE-2100
Precise count and classification of bone marrow cells is useful
for diagnosis and classification of diseases such as acute
leukemia and myelodysplastic syndromes as well as for the
monitoring of the effects of chemotherapy and bone marrow
transplantation. Automated quantitative analysis of bone marrow cellular composition by hematology analyzers, however,
has always been difficult [1–3]. A number of problems, in fact,
arise from difficulty in erythroblast identification, contamination by fat particles, heterogeneity of cell types and maturation levels, and the presence of low-frequency and very large
cells. In the hematology routine analyzers of the last generation technological advances have solved some of these difficulties, e.g. the identification of erythroblasts [4]. The XE-2100
automated blood cell counter can effectively count, besides
the physiological 5 leukocyte subtypes, erythroblasts and immature granulocytes using laser light flow cytometry and
DNA fluorochromes. In this study we have evaluated the performance of the XE-2100 for the analysis of bone marrow
cells.
40
20
0
Patients and Methods
b
Study Samples
All samples consisted of 1–2 ml of bone marrow fluid collected in K2EDTA and analyzed on the XE-2100 within 4 h of venipuncture. Before
aspiration samples were gently mixed by inversion. No filtration nor any
other treatment of samples was carried out. Microscope analysis was performed on May-Grünwald-Giemsa-stained bone marrow smears. A total
of 500 leukocytes per film was counted. Since the XE-2100 does not include NRBC count in the WBC count, all results were corrected to a total
nucleated cell count (TNCC) calculated as TNCC = NRBC + WBC.
278
Infus Ther Transfus Med 2001;28:277–279
20
40
Microscope
60
80
Fig. 1. Correlation of bone marrow cell counts obtained with the XE2100 and the microscope. a granulocyte percentage. b erythroblast percentage.
Results
Precision and Comparability
Reproducibility of the XE-2100 measurements was assessed
through duplicate analysis of 165 samples. Coefficients of variation were below 15% for all counts except erythroblasts and
lymphocytes. In 57 samples the instrument did not report a
complete differential count. We compared the proportions of
bone marrow granulocytes and erythroblasts by the XE-2100
with the microscope count using linear regression and analysis
of differences on 207 samples out of a total of 264 samples, in
which a complete computation was available. Correlation between XE-2100 and the microscope (fig. 1a) was excellent for
granulocyte percentage (r2 = 0.657). Analysis of differences
indicated a very good agreement in the great majority of samples, with a mean difference (XE-2100 – microscope) of
–0.9%. Correlation between XE-2100 and the microscope was
slightly lower for erythroblast percentage (r2 = 0.576) (fig. 1b),
with a mean difference (XE-2100 – microscope) of –13.4%.
There was a tendency of the XE-2100 to slightly underestimate erythroblast percentage in most of the samples.
Zini/Mistretta/Giordano/Laurenti/d’Onofrio
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The XE-2100 Counting Method
The XE-2100 hematology analyzer (Sysmex, Kobe, Japan) performs white
cell differential counts measuring i) radiofrequency and direct current resistance (indicators of cell content density and cell size), ii) forward and
side scatter light emitted by a semiconductor laser beam (indicators of
cell size and internal components), and iii) side fluorescence. Both mature
and immature granulocytes are identified and counted in the WBC channel: Moreover, immature granulocytes are identified in the immature
myeloid information (IMI) channel, owing to their partial resistance to
surfactant used in that channel. Nucleated red blood cell (NRBC) identification and counting is carried out after red blood cell lysis. Intact white
blood cells (WBCs) and NRBCs are stained with a fluorescent dye (in
Stromatolyser-NR). In the resulting two-dimensional cytogram the intensity of side fluorescence is represented on the x-axis, while the intensity of
the forward scattered light is represented on the y-axis. NRBCs are identified as a well-separated cell cluster to the left of the WBC population.
The NRBC count is expressed as a proportion per 100 WBCs and as absolute number per unit volume of blood. We have assessed the imprecision of the XE-2100 NRBC counting using linear regression and the
NCCLS-H20A method of differences between duplicates. Instrument
comparability has been assessed against 500 cell microscope counts using
linear regression and analysis of differences. The clinical usefulness of the
XE-2100 NRBC method has been assessed from observations of instrument reports in patients with hematological disorders.
0
samples in leukemia, hypoplasia or erythroid hyperplasia (fig.
2). Our study showed that analysis of bone marrow samples
with this method should be improved by an algorithm for fat
exclusion. A problem was the high percentage of samples with
incomplete differential count results (57/264): Dilution of the
samples with phosphate buffered saline (PBS) to counts less
than 20–30 × 109/l reduced the number of rejections and is
presently carried out routinely before analysis.
Discussion
Fig. 2. XE-2100 scattergrams: In the upper left scattergram (DIFF) the
y-axis represents the side fluorescence, the x-axis represents the side scatter. In the upper right scattergram (WBC/BASO) the y-axis represents
the forward scatter, the x-axis represents the side scatter. In the lower left
scattergram (IMI) the y-axis represents the alternating current signal
(radio frequency) the x-axis represents the direct current signal. In the
lower right scattergram (NRBC) the y-axis represents the forward scatter,
the x-axis represents the side fluorescence.
This example is obtained from a sample of bone marrow with normal cell
composition. The sigmoid clusters in the WBC/BASO and NRBC channel
are due to fat particles.
Clinical Usefulness
The TNCC was a good indicator of marrow cellularity. The
observation of instrument cell distribution plots (scattergrams) provided the morphologist with an immediate general
perception of the overall quality and characteristics of the
Previous attempts to analyze bone marrow samples with automated blood cell counters have obtained very limited results
[1–3], owing to difficulties such as the unpredictable behavior
of erythroblasts and interference by cellular debris and lipid
particles. The possibility to obtain rapid accurate cell counts
from bone marrow samples with the XE-2100, similar to that
reported with the Abbott CELL-DYN 4000 hematology analyzer [4], complements the morphological examination. Automated analysis permits a valuable objective preliminary
evaluation of marrow samples, prior to conventional microscope examination. It provides the morphologist with an immediate general perception of the overall quality and characteristics of the sample, the relative proportions of cells and fat,
total cellularity, myeloid/erythroid ratio, and presence and
characteristics of abnormal cell clusters. We could demonstrate a very good correlation between automated counts and
microscopic evaluation with respect to neutrophil percentage,
an acceptable correlation with respect to erythroblast percentages, and a consistently lower automated erythroblast count
compared to former published studies with flow cytometry
and CELL-DYN 4000 [5, 6]. Some problems still have to be
solved, such as interference by fat particles [4, 7], which often
falsely increase the total cell count, the presence of and interference by platelet clumps, the need to recalculate the TNCC
from WBC and NRBC counts, and finally the availability of
results only on research screens for a significant number of
samples.
References
Bone Marrow Analysis with Blood Cell
Counters
4 D’Onofrio G, Zini G, Tommasi M, Laurenti L,
Vergine C, Van Hove L: Quantitative bone marrow
analysis using the Abbott CELL-DYN 4000
hematology analyzer. Lab Hematol 1997,3:146–154.
5 Terstappen LWMM, Levin J: Bone marrow cell differential counts obtained by multidimensional flow
cytometry. Blood Cells 1992;18:311–323.
6 Sakamoto C, Yamane T, Ohta K, Hino M, Tsuda I,
Tatsumi N: Automated enumeration of cellular
composition in bone marrow aspirate with the CellDyn 4000 automated hematology analyzer. Acta
Haematol 1999,101:130–134.
7 Lesesve JF, Goupil JJ, Latger V, Buisine J,
Lecompte T: Artefactual elevation of the automated white cell count in the context of a bone marrow
aspirate analysis. Clin Lab Haematol 2000,22:56–
57.
Infus Ther Transfus Med 2001;28:277–279
279
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1 Tatsumi J, Tatsumi Y, Tatsumi N: Counting and differential of bone marrow cells by an electronic
method. Am J Clin Pathol 1986;86:50–55.
2 Bentley SA, Taylor MA, Killian DE, .Schoultz SB,
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