Science

Comparing Neubauer Hemacytometer, SY

Conventional, SY Located, and Automated Flow
Cytometer F–100 Methods for Urinalysis
Yung-Liang Chen, PhD,1 Mao-Kuei Chang,2 Yu-Jen Chen,1 Bao-Chy Chang3
(1Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsinchu, 2Shih-Yung Medical Instruments Co. Ltd,

Taipei, 3Department of Pathology and Laboratory Medicine, Armed Forces Shun-Shan Hospital, Taipei, Taiwan, Republic of China)
DOI: 10.1309/LM8POKTMIQXSSTHO

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Abstract
Background: The present study compared
various urinalysis methods for common
laboratory screening procedures.
Methods: The authors compared the Neubauer
hemacytometer, SY conventional, SY located,
and automated UF-100 flow cytometer
methods for the determination of red blood
cells (RBCs), white blood cells (WBCs), and
epithelial cells (ECs) in 182 subjects.
Results: Overall, each pair of the methods
were well correlated with a correlation value
r>0.9 (P<0.001) for RBC and EC using a linear
regression analysis. However, the r value for
WBC ranged between 0.73 and 0.98. Further
analysis, regardless of the method chosen,
reveals that the overall performance of WBC
count was slightly lower than that of RBC
and EC.
Conclusions: The data suggests that caution
is needed for the WBC measurement. We
also found that the UF-100 method tended
to overestimate the number of abnormal
individuals for all cell types relative to the
other methods. Since the flow cytometer
method is fast and less tedious, we suggest
that it is appropriate to use UF-100 for
conducting urinalysis in a large number of
patients, while the SY located method could
be used for a final confirmation.

Urine analysis for the presence of red blood cells (RBCs),
white blood cells (WBCs), and epithelial cells (ECs) remains
the most common screening procedure in laboratories for early
detection of renal or urinary tract disease in asymptomatic sub-
jects. The conventional procedure consists of using test strips
followed by a microscopic examination of urine sediment when
test strips are positive.1,2 This procedure, however, often gives a
significant rate of false-negatives or -positives when compared
with precise cell counts.3,4 Urine particle counting by visual mi-
croscopy is convenient, but is time consuming when examining
a large number of samples.5 Presently, there are 2 popular meth-
ods for cell counting: automated flow cytometer UF-100 and
manual SY located. The SY conventional method, originally
developed by Shih-Yung Medical Instruments, uses one kind
of grid slide with a 0.1-mm depth for cell count. The newly
developed SY located, however, uses 2 kinds of grid devices with
plurality of counting chambers, one with 81 square grids with
a 0.1-mm depth and the other with 24 rectangular grids with a
0.05-mm depth. The rectangular grids make the urine sediment
examination (containing blood cells and pus) easier and clearer
under microscopic observation.
Both the UF-100 and SY systems have claimed to be
precise and accurate for urinalysis. In the present study, we
evaluated these 2 methods and compared them with other
conventional measures such as the Neubauer hemacytometer
and SY conventional in order to offer a reference for clinical
laboratories.
Materials and Methods
Study Population
The study population consisted of 182 randomized sub-
jects who underwent physical examinations at the Taipei Jen-Ai
Municipal Hospital.
Urine Sample Preparation
More than 20 mL of fresh, midstream urine from each
subject was collected and examined within 1 to 2 h. Ten mL of
well-mixed urine was poured into the SY quantitative centrifu-
gal tube for cell count and hemacytometer analysis. The remain-
ing 10 mL was used for the other assay methods.
Automated UF-100 Flow Cytometry Analysis
In brief, after identifying each urine sample by its bar code,
the automated UF-100 analyzer (Sysmex) was used to aspirate
800 µL of the sample for cell, bacteria, and cast counts based
on electrical impedance for volume, forward light-scatter for
size, and fluorescent dye to determine nuclear and cytoplasmic
characteristics.6
Microscopy With the SY System
The SY system (Shih-Yung Medical Instruments, Taiwan)
consists of an SY quantitative centrifuge tube and an SY double-
grid counting slide 10. The centrifuge tube containing the urine
specimen was capped and centrifuged at 1,500 rpm for 5 min. Fol-
lowing aspiration of the supernatant to a marked level, the pellet
was agitated into a homogeneous mixture followed by a sampling
pipette provided by the manufacturer. After that, RBCs, WBCs,
and ECs were examined using a high-power field (HPF). The SY
double-grid counting slide 10 provides 2 types of grids for cell
counting. One is used for conventional cell counts (SY conven-
tional) and the other for locating the cells (SY located).
The conventional grid has 9 large squares that are evenly
divided into 9 small square grids (for a total of 81). Each large
square grid is 1 mm in length with a chamber depth of 0.10
mm. For cell counting, the diagonal small square grids (n=9
in each large square grid) were used for each cell type count at
a 400× magnification. The mean cell numbers per HPF were
calculated as below:
The mean cell numbers per HPF = n/9 × 1.6; where n is
the total number of cells counted in 9 small square grids.

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The new SY located, so-called “thinner platform,” con-
sists of 4 large square grids. Each grid is 1 mm in length with a
chamber depth of 0.05 mm. The large grid is further divided
into 6 small rectangular grids (for a total of 24). The volume of
each small rectangular grid is 0.0083 µL, which is equivalent to
the average volume of 1 microscopic HPF in the conventional
method of urinary sediment examination.
For urinary sediments, either cells per µL or cells per HPF
were reported. The 4 big square grids of the thinner platform
were used and counted at a 400× magnification. The mean cell
numbers per HPF were calculated as follows:
The mean cell numbers per HPF = N/12, while the mean
cell numbers per µL were calculated according to the mean cell
numbers per µL = N/4 × 1.2; where N is the total number of
cells counted in 4 big square grids.
Neubauer Hemacytometeric Analysis
The Neubauer hemacytometer contains 9 big square grids,
where the central big square is divided into 25 small square grids
and the surrounding ones are divided into 16 small square grids.
For cell counting, the diagonal small square grids, except the
middle ones (total 8 small square grids), were used and counted
at a 400× magnification. The mean cell numbers per HPF were
calculated as M × 2/9 × 1.6; where M is the total cell numbers
counted in 8 small square grids.
Statistical Analysis
The cell counts for RBCs, WBCs, and ECs of all 182
subjects were evaluated without knowing their clinical history.
Those subjects with cell counts greater than standard reference
values were defined as abnormal. The criteria for the reference
values of each cell count are given in Table 1. Percent of abnor-
mal is calculated by the number of subjects or cases (n) over the
investigated subjects, or as (n/182) × 100. Correlation between
each pair of methods was performed by scatter plot using a
linear regression in which a correlation coefficient (r) was estab-
lished. Levels of significant difference were determined by
a paired t test with P<0.05 as a cutoff.
Table 1_General Criteria for the Diagnosis in Subjects
With Normal and Abnormal Cell Ranges
Method Category Abnormal Range
Neubauer chamber RBC/HPF $3
WBC/HPF $5
EC/HPF >4
SY conventional method RBC/HPF $3
WBC/HPF $5
EC/HPF >4
SY located method RBC/HPF $3
WBC/HPF $5
EC/HPF >4
RBC/μL $12
WBC/μL $20
EC/μL >15
UF-100 RBC/HPF $3
WBC/HPF $5
EC/HPF >4
RBC/μL $17.6
WBC/μL $15.4
EC/μL >8.7
WBC, white blood cell; RBC, red blood cell; EC, epithelial cell; HPF, high power field.
228 LABMEDICINE j Volume 40 Number 4 j April 2009 labmedicine.com
To determine the effect of cell types on the counting per-
formance, the number of individuals whose cell-count values
being either false-negative or false-positive (determined by all
the methods combined) were summed. This number was then
defined as the cases that are not consistent. For instance, the
RBC values in a subject counted as “normal” by UF-100 but
“abnormal” by the others was classified as not consistent. The
remaining number of cases are defined as “consistent.” Percent
consistency was calculated by ([182–n]/182) × 100; where n =
number of cases that were not consistent. Therefore, each RBC,
WBC, or EC count possesses its own percent consistency.
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Results
Categorization
The criteria for defining the samples as normal and abnor-
mal are listed in Table 1. Individuals that show cell-count levels
greater than that of standard reference values used in the clini-
cal laboratories are considered to be abnormal, but they are not
necessarily associated with clinical symptoms.
Correlation Between the Methods
First, we compared the correlation between the methods
using linear regression for all 182 subjects regardless of normal
or abnormal values. Table 2 shows that each pair of methods
was significantly correlated with each other between every
2 methods for RBC and EC count with a correlation coef-
ficient (r) greater than 0.9 (P<0.001). The correlation was
slightly lower for WBC measurement but remains significant
(P<0.001). Also, the mean levels of RBC, EC, and WBC deter-
mined by each method were not statistically different with
a wide range of standard deviation (data not shown).
Identification of Abnormals Using Neubauer
Hemacytometer, SY Conventional, SY Located,
and Automated UF-100 Flow Cytometer
Since the abnormal values of patients are critical and es-
sential in clinical practice, we further evaluated the number
and percentage of abnormal subjects determined by each
method according to the criteria listed in Table 1. Table 3
shows that the percentages of abnormal patients identified
by Neubauer hemacytometer, SY conventional, SY located,
and UF-100 methods were 17.03%, 17.03%, 16.48%, and
21.98% for RBC count, respectively. Likewise, the percentages
were 12.09%, 12.64%, 12.09%, and 18.69% for EC and were
15.38%, 15.38%, 16.48%, and 26.37% for WBC. Most inter-
estingly, the number of abnormal subjects identified by auto-
mated UF-100 tended to be larger than the other methods,
particularly for the WBC measurement (Table 3).
Effect of Cell Types on the Overall Counting
Performance
We next evaluated the effect of cell types on the overall
counting performance. Those individuals whose cell-count
values in each respective cell type (for example, RBC) that
give either false-positives or -negatives judged by all 4 methods
were defined as “inconsistent” cases (please refer to the section
on statistics in Materials and Methods for definition). Table
4 shows that there were 171, 165, and 156 consistent cases
for RBC, EC, and WBC, respectively (containing no false-
 Science

Table 2_Correlation Between Each Pair of Methods for Red Blood Cell, Epithelial Cell, and White Blood Cell Count

Methods for Red Blood Cell

R Neubauer Counting Chamber UF-100 SY Located Method SY Conventional Method

Neubauer counting chamber – 0.91 0.99 0.97

UF-100 0.91 – 0.95 0.95
SY located method 0.99 0.95 – 0.98
SY conventional method 0.97 0.95 0.98 –

Methods for Epithelial Cell

R Neubauer Counting Chamber UF-100 SY Located Method SY Conventional Method

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Neubauer counting chamber – 0.93 0.95 0.96
UF-100 0.93 – 0.91 0.92
SY located method 0.95 0.91 – 0.92
SY conventional method 0.96 0.92 0.92 –

Methods for White Blood Cell

R Neubauer Counting Chamber UF-100 SY Located Method SY Conventional Method

Neubauer counting chamber – 0.97 0.86 0.76

UF-100 0.97 – 0.83 0.73
SY located method 0.86 0.83 – 0.98
SY conventional method 0.76 0.73 0.98 –
Correlation (r) between the 2 methods was determined by a linear regression analysis.
All P values were less than 0.001.

Table 3_Percent of Abnormal Subjects or Cases Determined by Each Analytic Method From 182 Subjects

% Abnormals ([n/182] x 100)

Method (n) Red Blood Cells (n) White Blood Cells (n) Epithelial Cells

Neubauer hemacytomer (31) 17.0 (29) 15.9 (22) 12.1

SY conventional method (30) 16.5 (24) 13.2 (24) 13.3
SY located method (31) 17.0 (30) 16.5 (22) 12.1
UF-100 (40) 22.0 (44) 24.2 (26) 14.3
* Abnormal individuals (n) are defined as cell count greater than standard reference values (see Table 1).

Table 4_Effect of Cell Types on the Overall Cell-Count Performance

Cell types Total Cases * Inconsistent Cases Consistent Cases % Consistency

Red blood cells 182 11 171 94

Epithelial cells 182 17 165 90.7
White blood cells 182 26 156 85.7
* Inconsistent cases represent for the number of subjects with either false-positives or -negatives determined by all methods combined (see Materials and Methods)

labmedicine.com April 2009 j Volume 40 Number 4 j LABMEDICINE 229

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negatives or -positives). The percentage of case consistency for
WBC (85.7%) seemed to be low relative to RBC (94%) and
EC (90.7%) (Table 4). This was because the automated UF-100
tended to overestimate WBC counts and lowered the overall
consistency. However, following the removal of those incon-
sistent cases, the correlation between each paired method for
WBC as well as for RBC and EC was highly correlated (r>0.9)
(data not shown). It appears that caution is needed when using
UF-100 for WBC analysis. The strategy in the use of UF-100
for urinalysis is discussed below.
Discussion
We evaluated and compared urinalyses of 182 subjects
using Neubauer hemacytometer, SY conventional, SY located,
and automated UF-100 methods. Each pair of methods in the
determination of RBC, WBC, or EC is significantly correlated
and acceptable. However, the abnormal cases determined by
UF-100 tended to be higher than the other methods (Table 3).
The reason for such overestimation (for example, RBC) has
been mainly due to the presence of crystals, yeast, sperm, and a
large number of bacteria as already pointed out by others.7,8 The
presence of naked EC nuclei may also contribute to the overes-
timation of WBC.9 Some other factors, such as the presence of
cast and mucus in the urine samples, should also be taken into
Gross Specimen Teaser No. 2
230 LABMEDICINE j Volume 40 Number 4 j April 2009 labmedicine.com
consideration as they may interfere with the diffusion of cells in
the SY double-grid counting slide 10.
Furthermore, based on the clinical diagnosis, it is neces-
sary to monitor the location of hematuria by examining the
morphology of urinary RBCs.10-13 Using a microscope, the
detection of abnormal shapes (such as acanthocytes and other
dysmorphic forms) as a marker for glomerular hematuria has
become a tradition in erythrocyte analysis.10,14 Nonetheless,
the different reference for populations, specimen collection
procedures, and analytical handling has to be carefully evaluated
prior to the determination by each method. Caution is needed
for the establishment of normal ranges in adults,15 infants, and
children.16 In addition, the number of cells found in the sample
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can be affected not only by the method of counting but also by
the test tubes used,1,7 to which cells, casts, and other particles
can adhere. Other mechanical forces, such as transportation and
centrifugation, can also damage the cells to be counted.
Taken together, the first 3 methods were conducted by
well-trained laboratory technicians using a high-power field
(HPF) to visually confirm the cell types, whereas the UF-100
is based on electrical impedance for forward light-scattering
and fluorescent staining. Therefore, the UF-100 method may
overestimate the cell count, which is unavoidable due to the
nonspecific fluorescent staining and interference of light scat-
tering by cell debris.

In conclusion, we agree that the UF-100 method is faster
and less tedious than the other methods, but it cannot provide
the detailed morphology of the target cells obtained by mi-
croscopic examination, and, consequently, it cannot replace
the conventional microscopic assessment.15,17–20 Since this
automated method tends to overestimate the cell counts, we
suggest that the UF-100 method could be used as an initial
high-throughput screening, while the SY system could be a con-
firmative approach in clinical practice. LM
Acknowledgments: We thank the Department of Pa-
thology and Laboratory Medicine of Taipei Jen-Ai Municipal
Hospital for technical assistance and providing the necessary
equipment and reagents for conducting this study. We also
especially thank Dr. Simon JT Mao and James Lee of National
Chiao Tung University, Taiwan, for the critical suggestions in
preparing the manuscript.
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