Discussion
A proper standardised method of urine microscopy provides both correct
identification of the different formed elements and quantifies them with
accuracy and precision. However, no such method exists in the real
world. Urine microscopy is a simple method to analyse cells and
sediments in the urine and is considered the ”gold standard”
technique.5 For particle counting, bright-field
microscopy of unstained preparations although performed regularly is not
always adequate for the detection of RBCs, bacteria, fungi and hyaline
casts. Therefore, there is a need for supravital staining phase-contrast
microscopy, or both for better examination of these urine sediments and
organisms. However, phase-contrast microscopy is not readily available
in all the labs. There are other preanalytical variables which affect
the quality of microscopic examination like the amount of urine
remaining in the tube for resuspension, speed and time of centrifugation
and whether the urine is either stained or not.
The microscopic examination performed manually is a labour-intensive and
time-consuming procedure with manual subjective variations and false
negative results. Additionally, it demands skilled personnel to ensure
precise outcomes and proper interpretation. To address these challenges,
there is the emergence of automated urine analyzers designed to
streamline the urine analysis process. 6,7
Automated urine analysis started with the use of inbuilt microscopes and
flow cytometry inspired by CBC analyzers. These have been developed for
high-workload laboratories to provide standardization and precision of
results as well as save time. By adopting strategic analysis methods
that leverage the unique strengths of various analyzers, valuable
benefits can be realized. These encompass not only staff labour savings
and enhanced testing efficiency but also a heightened standard of
testing quality.11
These automated urine analyzers are based on two analytical principles
for urine sediment analysis, one based on electrical impedance, and the
other on image-based analysis that sorts particles according to preset
particle dimensions. It is not known which principle is better than the
other. Automated urine sediment analyzers like UF-1000i urine analyzer
from Sysmex, Kobe, Japan works on Flow cytometry whereas Cobas 6500
urine analyzer from Roche Diagnostics Mannheim, Germany functions on the
assessment of digitalized images capture of formed elements in urine8,9 with great accuracy in detecting Red cells white
cells as well as epithelial cells .10
There could be cell destruction in the Manual microscopic examination
during processing and handling of the sample .FD Ince et al., concluded
that there is a good agreement between the automated analyzer and manual
microscopy for WBCs and epithelial cells. 11 good
agreement has been noticed between the manual method and Iris iQ200 for
red cells, white cells and epithelial cels.12,13 This
was similar to the observation made in our study.
However, the difficulty with automated Analyzer is that it does not
count damaged WBCs but it may count distorted and lysed cells as an
artefact. Shayanfar et al., in their study, concluded that Iris iQ200
counts fewer RBcs if there are presence of dysmorphic cells and ghost
cells in cases of hemolytic disorder or
glomerulonephritis.14 Similar false-positive results
have also been observed by Wah et al.12 Therefore
samples from the urine of patients suffering from renal disorders, like
glomerulonephritis need to be reconfirmed by manual microscopic
evaluation.12
In cases of hematuria in glomerulonephritis, hematuria, RBCs travel both
the glomerular filtration barrier and the convoluted tubules which can
cause changes in RBC morphology.15 If the red cell
gets lysed or becomes dysmorphic in the process, it might not be
identified through microscopic examination. The automated urine sediment
analyzers excel at identifying normal-shaped RBCs, but they might not
perform as well when it comes to detecting RBCs with abnormal
morphologies.16,17
There are some discrepancies also noted in the analysis of
microorganisms and a possible reason could be the inefficiency of the
software installed in classifying them.14 Falsely high
RBC count may occur due to misclassification of
yeasts.14 Chien et al., found differences in the
presence and count of bacteria in most samples by microscopy when
compared with another urine analyzer Iris iQ200.6Similarly, FD İnce et al., noticed the presence of bacteria in more
number of cases by manual microscopy when compared to the Automated
analyzers.8We also did not get any concordance in
microorganisms, crystals and casts similar to these studies.
The accuracy of Urine particle flow cytometers is better than microscopy
and is beneficial in saving time as well as human manpower with better
accuracy. 18 The initial urine flow cytometer in the
market was UF-100 (Sysmex, Kobe, Japan), which helped in identifying
squamous epithelial cells, transitional epithelial, tubular cells, red
cells, white cells, bacteria, fungus, hyaline casts, crystals, and
spermatozoa, using argon laser flow cytometry. It showed acceptable
linearity over clinically working ranges, and consistently and
significantly less imprecision was detected compared to manual
microscopic evaluation with a negligible carry-over. UFCs have been
compared with chamber counts, quantitative urine microscopy tests, test
strips, sediment counts, bacterial culture, and others and others showed
similar results .18-20 This technology is useful in
diagnosing and monitoring urinary tract infections and other
non-infectious renal disease.9
The newer variant of Sysmex analyzers( UF-5000 and UF-4000 ) works on
the principle of forward scatter light ,side scatter light , side
fluorescent light, and depolarized side scattered light .The newer DSS
technique is a unique technology which helps in detecting crystals and
differentiating them from red cells.28This can
significantly reduce the need for manual microscopy without affecting
the quality of the test .9
Flow cytometry has demonstrated its superiority in detecting both blood
cells and bacteria and is dependent upon the concentrations of different
elements within the sample. The utilization of image processing is
promising in enhancing detection capabilities, granting Lab
technologists/pathologist a chance to visualise distinct images.
Sysmex launched another variant UF-1000i which works on bacteria forward
scatter (FSC) and fluorescent light scatter (FLH) and differentiates
between gram-positive and gram-negative bacteria which causes urinary
infections.29 It can reduce the frequency of samples
needing culture, thereby causing ease in diagnosis time, and reducing
the time and costs of each test 23,24 This new
technology has been found to reduce the number of culture by
28–60%.25,26 This faster technique will
substantially reduce the need for unnecessary empirical antibiotic
therapy. However, a drawback of this autoanalyzer compared to urine
culture is that it counts both living and dead bacteria yielding a
higher bacterial count.
Enko et al. conducted a comparative study on 195 urine samples between
the UF-5000 (Sysmex , Kobe, Japan) working on UFC and the cobas® u 701
(Roche Diagnostics, Rotkreuz, Switzerland) urine analyzers which work on
manual phase-contrast microscopy. The level of agreement between the
UF-5000 and manual microscopy was nearly perfect (κ > 0.8)
for tubular epithelial cells, red blood cells, white blood cells,
hyaline casts, bacteria, and yeast. Cobas® u 701 analyzers ashowed
substantial agreement (κ = 0.61-0.80) for white blood cells, moderate
agreement (κ = 0.41-0.60) for hyaline casts, and fair agreement (κ =
0.21-0.40) for RBCs, squamous cells, tubular epithelial casts, bacteria,
and yeast. UF-5000 analyzer demonstrated a sensitivity ranging from
98.5% for red blood cells to 83.3% for casts. On the other hand, the
cobas® u 701 analyzer had a sensitivity ranging from 83.0% for white
blood cells to 31.6% for yeast cells. Thus they concluded that the
UF-5000 analyzer had a stronger agreement with manual microscopy
compared to cobas® u 701 modules and recommended the Sysmex urine
analyzers can be used with a reliable result. However, urine samples for
cast and crystals should be confirmed manually.27
Tantisaranon et al analyzed in 2021 and examined 100 routine urine
samples to compare three different analyzers– Cobas 6500, UN3000-111b,
and iRICELL 3000. They found there was a good correlation between the
three urine analyzers with an overall concordance of more than 80%. The
level of agreement between manual analysis and the three instruments for
sediment analysis varied. It was very good to good for erythrocytes,
leucocytes, and epithelial cells, and moderate for bacteria. Fair to
good agreements were found between manual microscopy and the 3
instruments for cast detection. They concluded that automated urine
analyzers could serve as effective tools for initial urine testing.
However, they also emphasized the necessity for manual microscopic
analysis to accurately classify urine sediments, particularly in cases
involving pathological specimens. The results were similar to our
study.28
The iQ200 analyzer ( Iris Diagnostics CA, USA ) works on the principle
of laminar flow digital imaging technology. It classifies and quantifies
cells and analyzes each particle based on its distinctive attributes,
such as shape, contrast, and texture. The operator has the option to
review and reclassify the generated images into their appropriate
categories.29-30 FD İnce et al ., found that there was
a fair agreement for yeast cell analysis between Iris iQ200 and the
manual microscopic method.11 Chien et al., postulated
that Yeast cells and crystals are not key elements for particle analysis
and could be removed by adjusting the corresponding thresholds in Iris
iQ200 reports.6
There is a strong correlation between the iQ200 output and manual
microscopic cell counts for epithelial cells, red cells and white blood
cells. However, it did not accurately count damaged WBCs and tended to
undercount RBCs when abnormal RBCs like ghost cells and dysmorphic cells
were present.31 Some issues arise during the analysis
of microorganisms.32It is challenging to classify as
cocci ”bacteria” although better results have been achieved for some
rod-shaped forms .32It can also detect malignant or
atypical urothelial cells, with a sensitivity rate of 87.5% for
identification. Shayanfar et al. observed that although casts were
detected by Iris iQ® 200 the instrument was not able to differentiate
between various casts.14 There were other studies
which observed that the automated machine was unable to detect the cast
by automated Analyzers.12 Therefore they recommended
that manual microscopic examination needs to be done for cases where
urinary cast is suspected. 11,14
There is a wide variation noted in the sensitivity and specificity of
the obtained results by different analyzers. These could be due to
variations in the clinical conditions of the patient populations
enrolled in these different studies. These could also be attributed to
the difference in definitions used to classify UTIs. Therefore, we
conclude that the applicability of flow cytometry strongly depends on
population characteristics to screen various cases.33,34
To streamline the laboratory work, automated analyzers need to be
successfully installed. In addition to mechanical integration, the
development of expert systems needs to be enabled and integrated to
successfully and accurately identify cases which need manual review,
improving the overall quality of the results.35
Limitations: The results may differ from other studies as the
data collected has a smaller sample size.
Conclusion: Manual microscopy although commonly used requires
well-trained and experienced staff and is time-consuming, especially in
a high throughput lab. We observed that the UF-5000 analyzer exhibited
stronger diagnostic agreement with manual phase-contrast microscopy
compared to the modules. Agreement with manual microscopy of the results
obtained with the Sysmex analyzers was reasonably good except in the
case of crystals, casts, and bacteria. Automated urine analyzers can be
used as a reliable option for urine sediment analysis. However, the
urine samples should still be confirmed through manual microscopy,
especially in cases of bacteria, fungus, cast and crystals. This can
save staff labour, make the testing more efficient, and ensure better
quality testing by adopting logical analysis strategies that exploit the
advantageous features of different analyzers.
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Microbiol 2010;48(9):3117-3121.doi:10.1128/JCM.00617-10.Legends 1a.Smears examined show singly scattered epithelial cells in urine
sediment smear. ( unstained slide, 400x.).The inset shows a squamous
epithelial cell in 100X ( unstained slide, 400x.)
1b. Smears examined show small clusters of round to oval tubular
epithelial cells along with a few pus cells(WBCS) in urine sediment
smear. ( unstained slide, 400x.)
1c. Smears examined show small singly scattered round to oval cells with
an absence of a nucleus (Red blood cells.) ( unstained slide, 400x.)
1d. Smears examined show singly scattered and small clusters of round to
oval with the presence of granules ( WBCS). ( unstained slide, 400x.)
2a. Smears examined show the presence of envelope-shaped oxalate
crystals marked with a blue arrow; ( unstained slide, 400x.)
2b. Smears examined show the presence of coffin lid-shaped triple
phosphate crystals marked with blue arrow; ( unstained slide, 400x.)
3a. Smears examined show thin rod-shaped bacilli, marked with blue
arrow;( unstained slide, 400x.)
3b. Smears examined show fungus with pseudohyphae, marked with blue
arrow; ( unstained slide, 400x.)
4a. Smears examined show few RBCs with hyaline cast; ( unstained slide,
400x.)
4b. Smears examined show few RBCs with hyaline cast; ( unstained slide,
400x.)
4c. Smears examined show few scattered RBCs with RBC Cast; ( unstained
slide, 400x.)
4d. Smears examined show few scattered WBCs with the presence of WBC
Cas; ( unstained slide, 400x.)