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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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.)