Automated flow cytometry compared with an automated dipstick reader for urinalysis.
The analytical performance and the accuracy of the OF-100 analyzer have been evaluated in detail by comparison with manual microscopy (12). The OF-100 generally performs accurate and precise quantitative urinalysis; however, detection of casts with the OF-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 OF-100 data with those of an automated strip-reader. For this purpose, a cross-check of OF-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
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).
The Sysmex OF-100 (TOA Medical Electronics Co.) uses argon laser flow cytometry. The OF-100 aspirates 800 [micro]L 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) (3)
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.
The manual microscopic sediment examination was performed according to the NCCLS guideline (14). After urinalysis with the OF-100, each urine specimen (10 mL) was centrifuged at 4008 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
OF-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 OF-100; negative for dipstick and microscopy), III (negative for OF-100; positive for dipstick and microscopy), or N (negative for all three systems) considering the cutoff value defined by the manufacturer (25 cells/[micro]L or 5 cells/HPF). Cases of discrepant dipstick and microscopic analysis were classified into groups Va (positive for OF-100 and microscopy; negative for dipstick) and Vb (negative for OF-100 and microscopy; positive for dipstick). Concordant OF-100 and dipstick results with a different microscopy result were classified into groups VIa (positive for OF-100 and dipstick; negative for microscopy) and VIb (negative for OF-100 and dipstick; positive for microscopy).
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.
A fairly good agreement was found between the OF-100 RBC count and the hemoglobin test strip reaction (Spearman r = 0.636; P <0.001; Fig. 1A). 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 OF-100. Among the group II cases, there were 4 samples with high crystalline content (uric acid) and 5 samples containing yeast cells.
[FIGURE 1 OMITTED]
The conductivity measured by the OF-100 correlated with the relative density of the urine (Spearman r = 0.541; P <0.001). Among samples with low urine conductivity (<5 m5/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 OF-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 [greater than or equal to] 8; classified in group VIa), OF-100 and dipstick RBC data were comparable, whereas lysed RBCs were not identified by manual microscopy.
To investigate the effect of vacuum sampling, OF-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 m5/cm), the OF-100 RBC count and the percentage of nonlysed RBCs were comparable between the two sampling methods. In contrast, the OF-100 RBC count and the percentage of nonlysed RBCs in three urine specimens with low conductivity (<5 m5/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 OF-100 WBC count and the leukocyte esterase test strip reaction (Spearman r = 0.785; P <0.001; Fig. 1B). 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 1). 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 [greater than or equal to] 8) and two samples with low conductivity (<5 m5/cm).
The OF-100 bacterial count differed significantly (P <0.0001) between nitrite-negative (median, 199 bacteria/ [micro]L; interquartile range, 92-458 bacteria/[micro]L; n = 875) and nitrite-positive urine samples (median, 956 bacteria/[micro]L; interquartile range, 417-2366 bacteria/[micro]L; n = 126). Among nitrite-positive samples, there were 18 cases with bacterial counts below the cutoff value defined by the manufacturer (250 bacteria/N,L). The OF-100 bacterial count correlated well with the OF-100 WBC count (Spearman r = 0.745; P <0.001; Fig. 2).
Effects of urine storage in vitro were investigated in 10 urine samples containing 87 bacteria/[micro]L (interquartile range, 57-183 bacteria/[micro]L) and 21 WBC/[micro]L (interquartile range, 18-27 WBC/[micro]L). Storage of these samples during 24 h at room temperature produced an increased OF-100 bacterial count (median, 491 bacteria/[micro]L; interquartile range, 306-872 bacteria/[micro]L; P <0.01). The OF-100 WBC count did not change significantly (median, 19 WBC/[micro]L; interquartile range, 14-25 WBC/[micro]L 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).
[FIGURE 2 OMITTED]
The OF-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 OF-100 cast and dipstick protein data. Microscopic sediment examination demonstrated the presence of casts in 73 urine samples, including 38 samples with hyaline casts ([greater than or equal to] 1 cast/LPF), 20 samples with pathological casts ([greater than or equal to] 1 cast/LPF), and 15 samples containing both types of casts. Among these samples, only 32 OF-100 hyaline cast counts and 14 OF-100 pathological cast counts were above the manufacturer-defined cutoff value (1 cast/[micro]L). False-negative OF-100 pathological cast counts (<1 cast/[micro]L; [greater than or equal to] 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),1.5g/L (n = 7),and 5.0 g/L (n = 5).
In a large number of cases, OF-100 hyaline cast counts (n = 123; 12.3%) and pathological cast counts (n = 81; 8.1%) were >1 cast/[micro]L, whereas microscopy detected <1 cast/LPF. Among the false-positive OF-100 pathological casts, we found urine samples with high WBC counts (>250 cells/[micro]L; 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 OF-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, OF-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 OF-100 was also compared with manual microscopy for squamous epithelial cells, spermatozoa, and yeast cells. The OF-100 performed well on epithelial cells and spermatozoa, showing only eight falsely increased epithelial cell counts (defined as >25 cells/[micro]L and <5 cells/ HPF; includes two urine samples with Trichomonas organisms), one false-positive sperm count (159 cells/[micro]L and 0 cells/HPF) in a sample with mucous threads, and no false negatives.
The OF-100 yeast cell count showed more discrepancies (6.9%). In 59 cases, OF-100 yeast cell counts were above the manufacturer-defined cutoff value (10 cells/ [micro]L), 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/[micro]L; n = 48). In 10 urine samples, OF-100 yeast cell counts were <10 cells/[micro]L, whereas >2 cells/HPF were detected by microscopy. Among these false negatives, we found five cases of falsely increased (group II) OF-100 RBC counts.
Other formed elements, such as oval fat bodies (n = 23) and Trichomonas organisms (n = 4), cannot be detected by the OF-100 instrument. Oval fat bodies were observed in the sediment from two urine samples with negative dipstick protein reaction; other protein test results were 0.258/L(n = 2), 0.75g/L (n = 4), 1.5g/L (n = 6),and 5.0 g/L (n = 9).
In this study, we compared Sysmex OF-100 urinalysis data with dipstick test results. Generally good agreement was obtained between OF-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 OF-100 instrument. Similarly, good agreement was obtained between the OF-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 OF-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 OF-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 OF-100 bacterial counts and OF-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 OF-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 OF-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 OF-100 RBC counts.
The detection of urinary casts by the OF-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 OF-100 cast counts. It is apparent that the OF-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 OF-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 OF-100 instrument (errors that may lead to an incorrect clinical interpretation of urinalysis). The low number of false negatives (4%)suggests that the OF-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 OF-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 OF-100 casts and yeast cells always require microscopic evaluation. An additional reduction of the error rate could be achieved by cross-checks of the OF-100 and dipstick data (RBCs vs hemoglobin, WBCs vs leukocyte esterase, casts vs protein, bacteria vs nitrite). Consequently, cross-checks of OF-100 and dipstick data could reduce the manual review rate. For example, high RBC and WBC counts raise flags on the OF-100 screen to review the specimen under a microscope. However, when OF-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 OF-100 instrument and theoretically would be missed in such a sieving system. However, Trichomonas organisms were found in some samples with false-positive OF-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 OF-100 sieving system may lead to a clinically acceptable urinalysis system. The OF-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.
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.
Received August 28, 1998; revision accepted November 2, 1998.
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(3) Nonstandard abbreviations: HPF, high-power field; LPF, low-power field; RBC, erythrocyte; and WBC, leukocyte.
MICHEL R. LANGLOIS,  JORIS R. DELANGHE,  * SOPHIA R. STEYAERT,  KAREL C. EVERAERT,  1riC~ MARL L. DE BUYZERE [I]
Departments of  Clinical Chemistry and  Urology, University Hospital Gent, De Pintelaan 185, B-9000 Gent, Belgium.
* Author for correspondence. Fax 32-9-2404985; e-mail email@example.com.
Table 1. Comparison of automated (UF-100), dipstick, and microscopic RBC and WBC counts. Group (a) UF-100 Dipstick Microscopy RBC, n (b) WBC, n (b) I + + + 450 444 II + - - 49 48 III - + + 9 2 IV - - - 456 482 Va + - + 2 2 (c) Vb - + - 5 (d) 0 VIa + + - 3 (e) 23 (f) VIb - - + 0 0 (a) RBC and WBC counts were cross-checked and classified into groups I to IV according to the manufacturer-defned cutoff value (25 cells/[micro]L or 5 cells/HPF). Groups II and III represent concordant dipstick and microscopy data with discrepant UF-100 counts; group V represents concordant UF-100 and microscopy data with different dipstick results; and group VI includes concordant UF-100 and dipstick data with different microscopy results. (b) Number of cases among 1001 urine samples. (c) Urine specimens with severe proteinuria (5.0 g/L). (d) Urine specimens with myoglobinuria (n = 2) and low conductivity (<5 mS/cm; n = 3). (e) Includes urine specimens with pH -8 (n = 9) and conductivity <5 mS/cm (n = 10). (f) Includes urine specimens with pH -8 (n = 3) and conductivity <5 mS/cm (n = 2).
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|Title Annotation:||Automation and Analytical Techniques|
|Author:||Langlois, Michel R.; Delanghe, Joris R.; Steyaert, Sophia, R.; Everaert, Karel C.; De Buyzere, Marc|
|Date:||Jan 1, 1999|
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