manual counting

Analytic performance of the ABX PENTRA DX 120
compared to the manual microscope method, the ADVIA 2120
and the SYSMEX XE 2100 in the cell counting of body fluids
P. Danise (1), B. Talento (2), A. Rovetti (1), C. Ricciardi (1), C. Esposito (2), A. Di Palma (1), D. Avino (1),
E. Tournier (3), M. Pastore (3), M.G. Pirofalo (1)
(1) Haematology Laboratory, Umberto I Hospital, Nocera Inferiore (SA), Italy
(2) Clinical Pathology Laboratory, Umberto I Hospital, Nocera Inferiore (SA), Italy – (3) Horiba Medical, Montpellier, France
Introduction
Results
Body fluids cell count is performed both in routine and emergency situations.
Often single laboratories lack dedicated specialists and therefore BF analysis
may be conducted by staff who do not have equal or necessary skill level to
do this diagnosis. Manual counting, that constitutes the basis of the reference
method, has an elevated analytic variability, by reason of the features of the
method itself and the limited number of counted cells. Such a method, used
in non standardized conditions and by personnel who perform a few numbers
of tests per year, may show reduced accuracy and reproducibility. In these
conditions the use of an automated counting system can be advantageous
in term of accuracy and reproducibility, as is the norm when an automated
procedure replaces a manual one.
Furtheremore we evaluated the possibility of counting cells distinct from
polymorphonuclear and mononuclear cells (neoplastic and mesothelial cells)
as a screening method for the subsequent correct microscope identification.
SYSMEX
XE 2100
Manual
count
4-5074
53-5207
1-67
5-6770
77-5199
1-42
5-5878
108-6122
1-76
10-5000
80-5000
1-60
Tab. 2 – Correlations of the total cell counts
ABX Pentra DX120
Advia 2120
SYSMEX XE 2100
SIEMENS
Advia 2120
SYSMEX
XE 2100
Manual
count
0.984
0.992
0.990
0.994
0.989
0.994
Ascitic F.
Pleuric F.
Cerebrospinal F.
MN
ABX
Pentra DX120
MN
Manual
PMN
ABX
Pentra DX120
PMN
Manual
OTHERS
ABX
Pentra DX120
OTHERS
Manual
26-99
21-99
0-100
3-86
20-99
0-100
1-74
1-79
0-100
14-97
1-80
0-100
6-86
4-80
0-50
10-85
7-82
0-13
Tab. 4 – Correlations of polymorphonuclear (PMN), mononuclear (MN),
and other (LIC) cells between ABX Pentra DX 120 and manual count
Ascitic F.
Pleuric F.
Cerebrospinal F.
Material & Methods
We examined 100 Body Fluid samples: 15 cerebrospinal (CSF), 42 pleural
and 43 ascitic fluids, coming from routine and emergency services at the
Hospital Central Laboratory. Total counting of each sample has been
conducted with all systems. Mononuclear, Polymorphonuclear and Large
Immature Cells (LIC) (OTHERS) counting, representing cells different from the
first two categories, has been performed only with the HORIBA Medical
system and the manual method.
Here the procedure specific to each apparatus:
(HORIBA Medical)
After cytochemical staining with chlorazol black, the cells are injected into
a double hydrodynamic focused cytometer that aligns cells and allows cell
by cell analysis by impedance (cell volume) and absorbance (cytoplasmic
and nuclear structure), through the determination of the diffraction of a
polychromatic light source following cell passage.
(SYSMEX)
The cells, after specific staining, are transported with a laminar flow into the
detection chamber where they cross a monochromatic light beam in a
hydrodynamic focused flow. Adapted optical systems (photodiodes and
photomultipliers) detect the diffused light at different angles of each cell.
The parameters measured by the optical bench in the Sysmex systems are
Forward Scattered Light and Side Scattered Light.
ADVIA 2120
SIEMENS
Advia 2120
(SIEMENS)
The total nucleate cell (TNC) counting in Ascitic and Pleural fluids is obtained
in the basophil/lobularity channel, after lysing of RBC and the leukocyte
cytoplasmic membrane, excluding basophils. For CSF specimen, previously
sphericized and fixed cells, are to be differentiated and counted by mean
of three optical signals (high-angle scatter, low-angle scatter and
absorbance); the signals are then converted into digital form.
MANUAL COUNTING
For fresh cell counting we used the Burker’s chamber.
For mononuclear, polymorphonuclear cells and LIC a slide was prepared
using Aerospray Hematology 7150® cytospin to deposit the cells. After
May Grumwald-Giemsa staining we performed microscope analysis.
MN
0.848
0.902
0.522 (P=0.06)
PMN
0.844
0.898
0.543 (P=0.05)
OTHERS
0.827
0.881
0.242 (P=0.38)
Conclusions
The total cell counting comparison between different analyzers showed a
good correlation of data without any relevant difference among the evaluated
technologies. Equally optimal results were obtained comparing the 3
instruments with the manual method. As expected, the cell number ranges
in the diverse body fluids showed values definitely lower in CSF.
This fact is a known cause of reduced accuracy within the performances of
the analyzers when faced with this kind of specimen. Indeed the
concentrations of cells are extremely lower in the CSF than in the blood for
which these technologies are meant.
The evaluation of polymorphonuclear, mononuclear, and other cells
performed in the comparison of the ABX Pentra DX 120 (HORIBA Medical)
and the manual method showed that:
(1) The correlation values, obtained for the pleural and ascitic fluids (Tab. 4),
make possible to consider this analyser for counting in routine use. This
is even truer if the results are correlated to the variance of the microscopic
counting performed by non specialized staff.
(2) The CSF correlation is less acceptable, due to the essentially very low
concentration of cells. Even in this case it is difficult to evaluate, without
further specific studies, what could be the variance of manual counting in
real life.
(3) The counting evaluation of cells different from polymorphonuclear
and mononuclear showed surprisingly positive results. The CSF was an
exception for the reason mentioned above. Such good performances
appear even more noteworthy if we consider the exclusive screening
role of the automated count that expects the manual microscopic
methodologies to produce the correct identification of the counted cells.
In conclusion automated body fluid counting has overcome the pioneer and
experimental phase, and it is now offering homogeneous instrumental
performances that can be a prelude to routine use.
The CSF cell count in automation presents nowadays aspects that have to
be improved, related to the low number of cells.
The comparison between the ABX Pentra DX 120, the other technologies
and the manual counting showed instrumental overlapping capabilities;
moreover it highlighted the possibility to identify polymorphonuclear and
mononuclear cells with satisfactory safety and to offer a reliable screening
method for the evaluation of the presence of other cells in the examined fluids.
REFERENCES
1.Marthe et al. “ Automated Flow Cytometric Analysis of Blood Cell in Cerebrospinal Fluid” Am J Clin Pathol 2004; 121: 690 – 700. 2.Harris et al. “ The ADVIA 2120 Hematology System: Flow Cytometry- Based Analysis of Blood and
Body Fluid in the routine hematology laboratory” Laboratory Hematology 2005; 11:47-61. 3.Brown et al. “ Validation of body fluid analysis on the Coulter lh 750” Laboratory Hematology 2003; 9: 155 – 159.
ISLH 2011
Here we present the comparison of a new counting mode proposed by
HORIBA Medical utilising Siemens’ and Sysmex’s technologies and the
manual method in order to evaluate the accuracy and the correlation between
the methods.
SYSMEX XE 2100
Ascitic F.
Pleuric F.
Cerebrospinal F.
HORIBA Medical
ABX Pentra DX120
Tab. 3 – Ranges (%) of polymorphonuclear (PMN), mononuclear (MN)
and other (LIC) cells
Aim
ABX PENTRA DX 120
Tab. 1 – Ranges of the total cell counts performed

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