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Clinical Chemistry 45: 118-122, 1999;
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(Clinical Chemistry. 1999;45:118-122.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Automated Flow Cytometry Compared with an Automated Dipstick Reader for Urinalysis

Michel R. Langlois1, Joris R. Delanghe1,a, Sophia R. Steyaert1, Karel C. Everaert2 and Marc L. De Buyzere1

Departments of
1 Clinical Chemistry and
2 Urology, University Hospital Gent, De Pintelaan 185, B-9000 Gent, Belgium.
a Author for correspondence. Fax 32-9-2404985; e-mail joris.delanghe{at}rug.ac.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recently, the Sysmex UF-100 flow cytometer was developed to automate urinalysis. We compared UF-100 test results with those of an automated dipstick reader. A cross-check of UF-100, dipstick, and microscopic sediment data was performed in 1001 urine samples. Good agreements (P <0.001) were obtained between UF-100 and dipstick data for erythrocytes (r = 0.636) and leukocytes (r = 0.785). Even in urine with low conductivity, the UF-100 could detect lysed erythrocytes. The UF-100 bacterial count was higher among nitrite-positive urine samples (P <0.0001) and was positively correlated with the UF-100 leukocyte count (r = 0.745; P <0.001). In stored urine (24 h), bacterial counts increased, whereas the forward light scatter of leukocytes decreased (P <0.01). Casts and yeast cells reported by the UF-100 should be confirmed by microscopic review because false positives occurred. We suggest that a computer-assisted cross-check of UF-100 and dipstick data allows a clinically acceptable sieving system to reduce the workload of microscopic sediment urinalysis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Microscopic urine sediment analysis has been the gold standard for examining urine cells and particles. However, it is imprecise and has wide interobserver variability (1). Moreover, it is labor-intensive and time-consuming. These problems have led to the widely used "sieve concept", which states that if the results of chemical screening with dipsticks are negative, then microscopic examination is unnecessary. Such simplification, however, may lead to substantial losses in diagnostic yield (2)(3)(4)(5)(6). Automation seems the answer to the need to improve both the accuracy and the productivity of urine sediment analysis (7)(8)(9)(10)(11). For this purpose, a flow cytometer-based walkaway instrument, Sysmex UF-100, that performs automated microscopic urinalysis was developed recently.

The analytical performance and the accuracy of the UF-100 analyzer have been evaluated in detail by comparison with manual microscopy (12). The UF-100 generally performs accurate and precise quantitative urinalysis; however, detection of casts with the UF-100 was found to be less reliable than the detection of cellular elements (12).

The feasibility of a flow cytometer-based "sediment sieve" for selecting samples that require microscopic examination remains unclear. In the present study, we explored the possibility of improving urine screening by comparing UF-100 data with those of an automated strip-reader. For this purpose, a cross-check of UF-100 data with results obtained by dipstick testing and microscopic sediment urinalysis was performed. Preanalytical and analytical factors such as urine concentration, sample storage, and use of evacuated sample containers were incorporated in the study.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patients and samples
We studied 1001 recently collected urine samples submitted for diagnostic urinalysis to our laboratory. The samples were obtained from patients (405 males, 596 females) at the Departments of Nephrology (n = 236), Urology (n = 69), Rehabilitation (n = 75), Intensive Care (n = 22), and other medical units (n = 599) in the University Hospital of Gent, Belgium. The majority of the samples were voided urine specimens (the midstream technique was recommended); others were sampled through a bladder catheter (n = 39), a self-adhering external catheter (n = 4), or a pyelostomy (n = 3). Urine samples from three patients who underwent a combined pancreas-kidney transplantation (with surgical drainage of the exocrine pancreas into the bladder) were obtained as well. The samples were collected in sterile containers, and 12-mL aliquots were transferred into test tubes and analyzed within 2 h. For additional preanalytical investigations, urine specimens from five patients with hematuria and three patients suffering from glomerulonephritis were aspirated (10 mL) into Uridraw vacuum test tubes (Terumo Europe).

sysmex uf-100
The Sysmex UF-100 (TOA Medical Electronics Co.) uses argon laser flow cytometry. The UF-100 aspirates 800 µL of uncentrifuged urine, dilutes the sample four times to dissolve the crystalline content, measures the urine conductivity, and analyzes the urinary formed elements by electrical impedance for volume, by forward light scatter for size, and by fluorescent dyes for DNA (phenanthridine) and membranes (carbocyanine). The pulse intensity and width of the forward scattered light and fluorescence light are measured. From these data, together with the impedance data, the urinary formed elements are categorized by multiparametric algorithms on the basis of their size, shape, volume, and staining characteristics. The results are displayed in scattergrams, histograms, and in counts per microliter as well as counts per high-power field (HPF).1

dipstick urinalysis
Dipstick urinalysis was carried out before flow cytometry analysis, using Combur 10-Test M strips and a Miditron automated reflectance photometer (Boehringer Mannheim) (13). The strips included reagent pads for semiquantitative assessment of relative density, pH, leukocyte esterase, nitrite, protein, glucose, ketones, urobilinogen, bilirubin, and hemoglobin/myoglobin.

microscopic urinalysis
The manual microscopic sediment examination was performed according to the NCCLS guideline (14). After urinalysis with the UF-100, each urine specimen (10 mL) was centrifuged at 400g for 5 min, and 9.5 mL of the supernatant was removed. In each specimen, at least 20 random microscopic fields were examined at x40 (HPF), and the mean number of cells or particles/HPF were calculated. Urinary casts were observed at x10 [low-power field (LPF)]. To reduce interobserver variability, all sediments were evaluated by the same experienced technologist.

classification of results
UF-100 and dipstick test results for erythrocytes (RBCs) and leukocytes (WBCs) were cross-checked and evaluated by manual microscopy. Results were classified into groups I (positive for all three test systems), II (positive for UF-100; negative for dipstick and microscopy), III (negative for UF-100; positive for dipstick and microscopy), or IV (negative for all three systems) considering the cutoff value defined by the manufacturer (25 cells/µL or 5 cells/HPF). Cases of discrepant dipstick and microscopic analysis were classified into groups Va (positive for UF-100 and microscopy; negative for dipstick) and Vb (negative for UF-100 and microscopy; positive for dipstick). Concordant UF-100 and dipstick results with a different microscopy result were classified into groups VIa (positive for UF-100 and dipstick; negative for microscopy) and VIb (negative for UF-100 and dipstick; positive for microscopy).

statistics
Data are presented as median and interquartile range. Statistical differences were evaluated using the Wilcoxon test. Agreement between automated cell counts and semiquantitative dipstick data was examined by Spearman rank analysis. P <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
erythrocytes
A fairly good agreement was found between the UF-100 RBC count and the hemoglobin test strip reaction (Spearman r = 0.636; P <0.001; Fig. 1 A). There were 65 cases (6.5%) of disagreement in the RBC count between the two methods, with 32 cases (3.2%) occurring near the cutoff value (Table 1 ). Two cases of disagreement were because patients had severe myoglobinuria (crush syndrome) and were classified in group Vb. Microscopic examination demonstrated that the majority of the discrepancies (49 group II cases) were related to overestimation of the RBC count by the UF-100. Among the group II cases, there were 4 samples with high crystalline content (uric acid) and 5 samples containing yeast cells.



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  		Figure 1. Box-and-whisker plots comparing flow cytometry (UF-100) and dipstick (Miditron) results for RBCs (A) and WBCs (B).

Significant agreement (P <0.001) was obtained by Spearman rank analysis for RBCs (r = 0.636) and WBCs (r = 0.785). The Miditron reports semiquantitative dipstick data as 0, 10, 25, 50, 150, or 250 cells/µL for RBCs and 0, 25, 100, or 500 cells/µL for WBCs. Boxes show medians and quartiles; whiskers are 10th and 90th percentiles of the representative UF-100 counts.


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  		Table 1. Comparison of automated (UF-100), dipstick, and microscopic RBC and WBC counts.

The conductivity measured by the UF-100 correlated with the relative density of the urine (Spearman r = 0.541; P <0.001). Among samples with low urine conductivity (<5 mS/cm; n = 15), there were only two group III cases and three group Vb cases (underestimation of RBC count because of lysis of RBCs). The UF-100 identifies lysed RBCs (ghost cells), which appear in the area of low forward light scatter in the RBC scattergram cluster. Even in nine urine specimens with alkaline pH (pH >=8; classified in group VIa), UF-100 and dipstick RBC data were comparable, whereas lysed RBCs were not identified by manual microscopy.

To investigate the effect of vacuum sampling, UF-100 RBC counts in five urine specimens from patients with hematuria were compared between conventional and vacuum test tubes. In two urine samples with normal conductivity (13 and 16 mS/cm), the UF-100 RBC count and the percentage of nonlysed RBCs were comparable between the two sampling methods. In contrast, the UF-100 RBC count and the percentage of nonlysed RBCs in three urine specimens with low conductivity (<5 mS/cm) were lower in vacuum tubes than in conventional tubes (at least 20% and 31% reduction, respectively), whereas hemoglobin dipstick reactions were comparable.

leukocytes and bacteria
A good agreement was obtained between the UF-100 WBC count and the leukocyte esterase test strip reaction (Spearman r = 0.785; P <0.001; Fig. 1BUp ). Discrepancies between the automated WBC count and leukocyte esterase were observed as well, and included 48 group II cases (4.8%) and only 2 group III cases (Table 1Up ). Group Va cases (n = 2) were associated with severe proteinuria (dipstick protein, 5.0 g/L). Among group VIa cases, we found three urine specimens with alkaline pH (pH >=8) and two samples with low conductivity (<5 mS/cm).

The UF-100 bacterial count differed significantly (P <0.0001) between nitrite-negative (median, 199 bacteria/µL; interquartile range, 92–458 bacteria/µL; n = 875) and nitrite-positive urine samples (median, 956 bacteria/µL; interquartile range, 417-2366 bacteria/µL; n = 126). Among nitrite-positive samples, there were 18 cases with bacterial counts below the cutoff value defined by the manufacturer (250 bacteria/µL). The UF-100 bacterial count correlated well with the UF-100 WBC count (Spearman r = 0.745; P <0.001; Fig. 2 ).



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  		Figure 2. Correlation between bacterial (y) and leukocyte counts (x) of the UF-100 instrument (Spearman r = 0.745; P <0.001).

Shown are data points ({square}; n = 1001), the linear regression curve (heavy solid line), and the 95% confidence intervals (thin solid lines).

Effects of urine storage in vitro were investigated in 10 urine samples containing 87 bacteria/µL (interquartile range, 57–183 bacteria/µL) and 21 WBC/µL (interquartile range, 18–27 WBC/µL). Storage of these samples during 24 h at room temperature produced an increased UF-100 bacterial count (median, 491 bacteria/µL; interquartile range, 306–872 bacteria/µL; P <0.01). The UF-100 WBC count did not change significantly (median, 19 WBC/µL; interquartile range, 14–25 WBC/µL after 24 h); however, the mean forward scattered-light channel of the WBC histogram was reduced from 87.0 (range, 80.1–102.3) to 64.5 (interquartile range, 58.2–76.1) after 24 h (P <0.01).

casts
The UF-100 discriminates between hyaline casts (without inclusions) and pathological casts (containing granular, cellular, or other inclusions that generate a fluorescence signal). No significant agreement was obtained between UF-100 cast and dipstick protein data. Microscopic sediment examination demonstrated the presence of casts in 73 urine samples, including 38 samples with hyaline casts (>=1 cast/LPF), 20 samples with pathological casts (>=1 cast/LPF), and 15 samples containing both types of casts. Among these samples, only 32 UF-100 hyaline cast counts and 14 UF-100 pathological cast counts were above the manufacturer-defined cutoff value (1 cast/µL). False-negative UF-100 pathological cast counts (<1 cast/µL; >=1 cast/LPF) were found in 21 urine samples. Among these false-negative counts, dipstick protein reactions were reported as negative (n = 1) or as 0.25 g/L (n = 2), 0.75 g/L (n = 6), 1.5 g/L (n = 7), and 5.0 g/L (n = 5).

In a large number of cases, UF-100 hyaline cast counts (n = 123; 12.3%) and pathological cast counts (n = 81; 8.1%) were >1 cast/µL, whereas microscopy detected <1 cast/LPF. Among the false-positive UF-100 pathological casts, we found urine samples with high WBC counts (>250 cells/µL; n = 63), high crystalline content (n = 2), mucous threads (n = 4), Trichomonas organisms (n = 3), and three samples from pancreaticocystostomy patients (with extremely high numbers of urothelial cells because of bladder irritation by proteolytic pancreatic enzymes) (15).

The effect of vacuum sampling on UF-100 cast counts was studied in urine specimens from three patients with glomerulonephritis. Hyaline and pathological cast counts measured in vacuum test tubes showed a marked reduction (at least 58% and 51%, respectively) compared with conventional tubes. Remarkably, UF-100 RBC counts in these urine specimens were higher in vacuum tubes than in conventional tubes (at least a 25% increase).

other formed elements
The UF-100 was also compared with manual microscopy for squamous epithelial cells, spermatozoa, and yeast cells. The UF-100 performed well on epithelial cells and spermatozoa, showing only eight falsely increased epithelial cell counts (defined as >25 cells/µL and <5 cells/HPF; includes two urine samples with Trichomonas organisms), one false-positive sperm count (159 cells/µL and 0 cells/HPF) in a sample with mucous threads, and no false negatives.

The UF-100 yeast cell count showed more discrepancies (6.9%). In 59 cases, UF-100 yeast cell counts were above the manufacturer-defined cutoff value (10 cells/µL), whereas <2 cells/HPF were detected microscopically. Among these cases, we found one sample that contained Trichomonas organisms and two samples that contained oval fat bodies; however, the majority was associated with the presence of high WBC counts (>250 cells/µL; n = 48). In 10 urine samples, UF-100 yeast cell counts were <10 cells/µL, whereas >2 cells/HPF were detected by microscopy. Among these false negatives, we found five cases of falsely increased (group II) UF-100 RBC counts.

Other formed elements, such as oval fat bodies (n = 23) and Trichomonas organisms (n = 4), cannot be detected by the UF-100 instrument. Oval fat bodies were observed in the sediment from two urine samples with negative dipstick protein reaction; other protein test results were 0.25 g/L (n = 2), 0.75 g/L (n = 4), 1.5 g/L (n = 6), and 5.0 g/L (n = 9).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we compared Sysmex UF-100 urinalysis data with dipstick test results. Generally good agreement was obtained between UF-100 RBC counts and the dipstick hemoglobin test. Even in urine samples with alkaline pH (14) or with low relative density and conductivity (16), the lysed RBCs were still detected by the UF-100 instrument. Similarly, good agreement was obtained between the UF-100 WBC count and the leukocyte esterase reaction, although the presence of esterase inhibitors in urine and severe proteinuria might negatively affect test results for leukocyte esterase (17)(18).

In a few cases, the UF-100 analyzer detected more RBCs and WBCs than did dipstick testing. Microscopic sediment analysis suggested that the majority of these cases involved overestimation by the UF-100 instrument. However, comparison with manual microscopy, the gold standard, is difficult because the latter technique has several methodological steps that may contribute to imprecision and inaccuracy (1), including centrifugation and resuspension steps that are either incomplete or lead to cellular loss and lysis.

The correlation between UF-100 bacterial counts and UF-100 WBC counts is of interest. The simultaneous analysis of these indicators allows the detection of preanalytical errors and hence better discrimination between urinary tract infection and growth of commensal bacteria. The increase in the bacterial count during sample storage is accompanied by a marked decrease of the WBC forward light scatter, whereas the UF-100 WBC count remains stable. The low WBC forward scatter can be explained by cell volume changes and suggests the presence of dead or aged leukocytes in urine stored for a long period of time after collection. However, these changes can also occur in vivo during a prolonged stay of WBCs in the bladder (19).

Vacuum urine sampling affects UF-100 test results for RBCs (only in urine with low conductivity) and casts, probably because of mechanical damage to these elements during aspiration. Disintegration of pathological casts during vacuum sampling causes a release of their cellular inclusions, as evidenced by increased UF-100 RBC counts.

The detection of urinary casts by the UF-100 is less definitive than is the detection of RBCs and WBCs. Similar findings have been reported by Ben-Ezra et al. (12). Comparison with manual microscopy demonstrated a high number of false-positive UF-100 cast counts. It is apparent that the UF-100 detects other formed elements as casts. We therefore recommend a manual review of those samples in which pathological numbers of urinary casts are found by the UF-100. However, some of these cases probably represent true detection of casts not identified by manual microscopy, which may occur in urine sediments with very large quantities of leukocytes.

In addition, the flow cytometric detection of yeasts is not always definitive. In several cases, the instrument had problems differentiating RBCs and yeast cells. This can be explained by a positive interference caused by yeast cells overlapping the RBC area of the scattergram. Dipstick testing (hemoglobin) may prove to be very useful in these cases.

Different mistakes were often found within the same sample. When we combined all cross-checked results of clinically relevant urinary formed elements, we calculated that 28% of the urine samples were not analyzed correctly by the UF-100 instrument (errors that may lead to an incorrect clinical interpretation of urinalysis). The low number of false negatives (4%) suggests that the UF-100 is suitable for use as a screening tool, but the number of false positives should be reduced by manual review (light microscopy). Therefore, criteria how to use the UF-100 as a sediment sieve are needed.

The use of UF-100-based decision rules could reduce the error rate of the instrument by manual review. In particular, positive UF-100 casts and yeast cells always require microscopic evaluation. An additional reduction of the error rate could be achieved by cross-checks of the UF-100 and dipstick data (RBCs vs hemoglobin, WBCs vs leukocyte esterase, casts vs protein, bacteria vs nitrite). Consequently, cross-checks of UF-100 and dipstick data could reduce the manual review rate. For example, high RBC and WBC counts raise flags on the UF-100 screen to review the specimen under a microscope. However, when UF-100 RBC and WBC data are concordant with dipstick hemoglobin and leukocyte esterase reactions, there would be no need for additional microscopic confirmation. For optimal use, we suggest that computer-assisted decision making is the optimal solution for sieving the urine samples.

Oval fat bodies and Trichomonas organisms cannot be detected by the UF-100 instrument and theoretically would be missed in such a sieving system. However, Trichomonas organisms were found in some samples with false-positive UF-100 casts (which cause review flags to appear on the screen) and would be detected by microscopic review of these samples.

In conclusion, dipstick testing combined with a computer-assisted UF-100 sieving system may lead to a clinically acceptable urinalysis system. The UF-100 analyzer is not a substitute for microscopic sediment examination; however, (when combined with dipstick testing) it can improve the productivity of urinalysis by reducing the numbers of specimens submitted to microscopy.


   Acknowledgments
 
We thank Sysmex (Toa Medical Electronics), Boehringer Mannheim, and Terumo Europe for providing the necessary equipment and reagents for conducting this study. We also thank G. Claeys (Department of Microbiology, University Hospital Gent) for helpful discussions.


   Footnotes
 
1 Nonstandard abbreviations: HPF, high-power field; LPF, low-power field; RBC, erythrocyte; and WBC, leukocyte.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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