Urea

Design Verification
Urea liquicolor
Contents
1
Introduction .................................................................................................................................................................. 2
2
Imprecision .................................................................................................................................................................... 2
3
Linearity and Detection Limit ...................................................................................................................................... 2
Linearity......................................................................................................................................................................................................... 2
Detection Limit ........................................................................................................................................................................................... 3
4
Recovery of Control Sera .............................................................................................................................................. 3
5
Comparison of Methods ............................................................................................................................................... 4
6
Interferences ................................................................................................................................................................. 6
7
Stability.......................................................................................................................................................................... 8
Recovery in Control Sera ......................................................................................................................................................................... 8
Linearity......................................................................................................................................................................................................... 8
Temperature Stress Results ................................................................................................................................................................... 8
8
Stability Working Reagent ........................................................................................................................................... 9
Recovery in Control Sera ......................................................................................................................................................................... 9
Linearity......................................................................................................................................................................................................... 9
9
Traceability .................................................................................................................................................................... 9
Form: 4.2-01.06-04
Rev. 009 | valid of 25.11.2020
1/9
1 Introduction
The imprecision and the recovery of the UREA liquicolor test has been tested and documented in order to verify its
clinical usefulness and additional features such as linearity, interferences and method comparison.
2 Imprecision
The imprecision (within-run and day-to-day) of the UREA liquicolor method was calculated from six determinations
on six consecutive days. Three pooled control sera were employed as sample material.
Analyser: HITACHI 717
Analyte concentration
(mg/dl)
27.3
63.3
276
Intra-assay
SD (mg/dl)
1.042
1.464
3.51
Inter-assay
%CV
3.82
2.31
1.27
SD (mg/dl)
1.115
2.557
11.157
%CV
4.08
4.04
4.04
3 Linearity and Detection Limit
Linearity
The linearity of the UREA liquicolor method was controlled by employing dilutions of a patient serum pool which
has been spiked with urea to approx. 450 mg/dl. The analysed concentrations were calculated vs. the regression
line. The deviation between analytical and regressed data are expressed in absolute and relative values. As an
example data measured on the Hitachi 717 are reported.
High Pool
Content (%)
0
10
20
30
40
50
60
70
80
90
100
Analytical Data
mg/dl
1.70
43.7
84.7
124
168
216
253
303
338
380
420
Design Verification and Product Data for UREA LIQUICOLOR
Rev. 009
Regressed Data
mg/dl
0.14
42.7
85.2
128
170
213
255
298
340
383
426
Deviation from Regression Line
mg/dl
(%)
1.56
1076.92
1.01
2.38
-0.53
-0.62
-3.62
-2.83
-1.91
-1.12
2.70
1.27
-1.89
-0.74
5.06
1.70
-2.38
-0.70
-3.22
-0.84
-5.56
-1.31
2/9
LINEARITY
35
Linear Regression
Error Bars = ± 2SD
Measured Analyte Concentration (mg/dl)
30
25
20
15
10
5
0
0
20
40
60
80
100
120
High Pool Content (%)
Conclusion:
The UREA liquicolor test is linear up to an urea concentration of at least 400 mg/dl.
Detection Limit
A 20-fold determination of a ‘0’ sample (phys. saline) on a Hitachi 717 analyser revealed an absolute mean of
-0.65mg/dl and a SD of 1.47mg/dl. From the three-fold standard deviation the detection limit can therefore be
calculated on the base of mean +3 SD to 5.06mg/dl.
4 Recovery of Control Sera
A number of commercially available control sera have been employed. The control sera have been
reconstituted/prepared according to the manufacturer’s instructions. Five-fold determinations of each control
serum have been performed with UREA liquicolor reagents of different production lots. The mean values have been
calculated and compared with the target values of the respective control sera.
CONTROL SERUM RECOVERY AT 578 nm
Control
LOT
serum
Fresh
Reference
10 days 37°C
12 days 37°C
Target
Range
Result
Deviation
Result
Deviation
Result
Deviation
Result
Deviation
mg/dl
mg/dl
mg/dl
(%)
mg/dl
(%)
mg/dl
(%)
mg/dl
(%)
-8.14
HumaTrol N
# 018
51.1
38.9 – 63.5
44.2
-13.46
41.3
-19.22
45.5
-10.96
46.9
HumaTrol P
# 016
172.0
131 – 213
157.2
-8.56
149.4
-13.12
469.3
-1.57
174.5
1.48
SERODOS
# 6868
52.
39.5 – 64.5
54.1
4.08
52.1
0.23
57.3
10.27
59.3
14.08
SERODOS
# 6795
172.7
131.3 –
157.3
-8.91
152.8
-11.52
166.7
-3.50
174.8
1.22
plus
214.1
Precinorm
# 154916
43.2
32.7 – 53.7
45.3
4.91
43.3
0.19
43.9
1.71
45.1
4.49
Precipath
# 199459
140
107 - 173
152.5
8.91
146.4
4.60
150.7
7.64
155.6
11.16
Summary
Conclusion:
610.7
585.36
633.44
656.4
No significant differences could be observed between fresh and stressed reagents, confirming
the stability of the reagents. Controls were all recovered well within the specified ranges.
As an alternative measurement wavelength, control recovery of fresh material was also assayed at 546nm. The
mean values of two determinations each were calculated and compared to the reference range of the respective
control sera. The corresponding recoveries at 578 nm were also measured for comparison.
Design Verification and Product Data for UREA LIQUICOLOR
Rev. 009
3/9
CONTROL SERUM RECOVERY
Control
LOT
serum
Fresh at 546 nm
Fresh at 578 nm
Target
Range
Result
Deviation
Result
Deviation
mg/dl
mg/dl
mg/dl
(%)
mg/dl
(%)
HumaTrol N
# 001
97.2
75.8 – 119.0
85.3
- 12.2
87.3
- 10.2
HumaTrol P
# 001
281.0
219 – 343
262.8
- 6.5
276.1
- 1.7
SERODOS
# 001
88.3
68.9 – 108.0
81.5
- 7.7
80.7
- 8.6
SERODOS plus
# 6798
143.0
112 - 174
140.1
- 2.0
143.6
0.4
Precinorm
# 150403
43.8
37.2 – 50.4
39.3
- 10.3
39.0
- 11.0
Precipath
# 150410
150
126- 174
139.9
- 6.7
143.1
- 4.6
748.9
- 6.8
769.8
- 4.2
Sum
803.3
Conclusion: All control sera were found within the respective reference ranges. Therefore, measurement at 546 nm
is feasible.
5 Comparison of Methods
The UREA liquicolor test has been compared against a commercially available urea method. Control sera as well as
patient samples (serum and urine) have been employed in the comparison.
The results have been evaluated by a main component analysis. The linear regression obtained could be described
as follows:
Serum:
N=
r=
Y=
Xmean =
Ymean =
Sample Reference
no.
1
UREA liquiUV
1
27.1
2
24.9
3
52.5
4
22.8
5
35.9
6
38.2
7
48.5
8
53.1
9
25.4
10
41.8
11
17.8
12
21.0
13
25.6
14
28.3
15
53.6
16
35.9
17
40.0
18
31.0
19
44.5
20
23.8
21
25.0
22
37.2
23
25.7
24
38.4
25
33.6
26
40.1
27
28.9
28
23.1
29
32.9
30
23.0
31
42.6
32
55.5
33
53.8
Test
2
26.5
24.0
53.2
23.3
36.1
39.2
48.6
52.9
25.4
43.1
18.5
21.0
26.4
26.2
52.9
35.9
39.4
30.0
44.1
23.0
26.4
35.7
25.4
40.1
36.0
41.7
29.0
22.2
33.2
23.0
40.2
54.3
52.9
1
UREA liquicolor
27.5
24.8
52.1
23.1
35.9
37.7
46.1
51.4
25.1
41.2
17.8
20.9
25.1
26.7
51.8
34.6
37.9
29.3
44.3
22.8
25.1
35.0
25.6
39.0
34.6
39.4
28.1
22.4
32.7
23.8
42.1
56.2
54.2
Design Verification and Product Data for UREA LIQUICOLOR
Rev. 009
55
0.998
0.981 X + 0.405
35.53
35.27
Mean
Reference
2
28.0
24.5
53.5
23.3
36.4
38.0
47.4
50.8
25.1
41.5
17.7
21.2
25.1
26.1
51.6
34.2
38.9
28.8
44.5
22.9
24.5
35.8
24.3
38.6
35.2
40.2
28.8
22.2
32.4
23.5
41.8
54.9
53.8
26.8
24.45
52.85
23.05
36
38.7
48.55
53
25.4
42.45
18.15
21
26
27.25
53.25
35.9
39.7
30.5
44.3
23.4
25.7
36.45
25.55
39.25
34.8
40.9
28.95
22.65
33.05
23
41.4
54.9
53.35
Test
27.75
24.65
52.8
23.2
36.15
37.85
46.75
51.1
25.1
41.35
17.75
21.05
25.1
26.4
51.7
34.4
38.4
29.05
44.4
22.85
24.8
35.4
24.95
38.8
34.9
39.8
28.45
22.3
32.55
23.65
41.95
55.55
54
Regression
26.70
24.40
52.26
23.02
35.73
38.38
48.04
52.41
25.33
42.06
18.21
21.01
25.92
27.14
52.66
35.63
39.36
30.33
43.87
23.37
25.62
36.17
25.48
38.92
34.55
40.54
28.81
22.63
32.84
22.97
41.03
54.28
52.75
Deviation
0.95
0.2
-0.05
0.15
0.15
-0.85
-1.8
-1.9
-0.3
-1.1
-0.4
0.05
-0.9
-0.85
-1.55
-1.5
-1.3
-1.45
0.1
-0.55
-0.9
-1.05
-0.6
-0.45
0.1
-1.1
-0.5
-0.35
-0.5
0.65
0.55
0.65
0.65
4/9
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
24.9
44.5
23.5
20.9
72.2
42.0
72.5
46.1
28.1
23.1
22.4
24.6
27.5
33.0
24.1
31.2
37.2
51.8
34.1
33.7
36.9
52.3
24.4
42.2
22.7
20.9
71.4
42.2
71.4
44.2
26.7
22.8
22.9
23.5
27.7
35.8
23.4
32.0
34.8
52.3
35.6
33.4
36.5
53.2
25.4
44.6
24.1
22.0
70.7
43.4
70.3
45.1
27.1
23.2
23.0
23.6
28.2
33.8
23.2
31.4
38.0
51.8
33.5
32.1
37.8
52.1
25.7
44.4
24.0
21.6
70.5
42.7
71.9
44.5
26.9
22.6
23.6
23.8
28.4
34.2
24.4
30.7
38.7
51.7
34.0
32.2
37.4
51.1
24.65
43.35
23.1
20.9
71.8
42.1
71.95
45.15
27.4
22.95
22.65
24.05
27.6
34.4
23.75
31.6
36
52.05
34.85
33.55
36.7
52.75
25.55
44.5
24.05
21.8
70.6
43.05
71.1
44.8
27
22.9
23.3
23.7
28.3
34
23.8
31.05
38.35
51.75
33.75
32.15
37.6
51.6
24.59
42.94
23.07
20.91
70.86
41.72
71.01
44.71
27.29
22.92
22.63
24.00
27.49
34.16
23.71
31.41
35.73
51.48
34.60
33.33
36.42
52.17
0.9
1.15
0.95
0.9
-1.2
0.95
-0.85
-0.35
-0.4
-0.05
0.65
-0.35
0.7
-0.4
0.05
-0.55
2.35
-0.3
-1.1
-1.4
0.9
-1.15
UREA liquicolor Serum
80
70
Test mg/dl
60
50
40
30
20
10
10
20
30
40
50
60
70
80
Reference mg/dl
Urine:
N=
r=
Y=
Xmean =
Ymean =
Sample Reference
no.
1
UREA liquiUV
1
2091
2
2889
3
818
4
1626
5
1131
6
1212
7
1131
8
1980
9
2454
10
2454
11
1242
12
1919
13
1768
14
970
15
616
Test
2
2050
2838
818
1646
1293
1242
1141
2000
2464
2424
1252
1919
1727
1000
586
1
UREA liquicolor
2104
2761
903
1630
1347
1286
1165
1943
2337
2387
1286
1943
1741
963
670
Design Verification and Product Data for UREA LIQUICOLOR
Rev. 009
38
0.995
0.974 X + 12.428
1451.08
1425.84
Mean
Reference
2
2054
2842
812
1610
1317
1175
1195
1842
2549
2448
1195
1781
1700
903
660
2071
2863
818
1636
1212
1227
1136
1990
2459
2439
1247
1919
1747
985
601
Test
2079
2801
857
1620
1332
1231
1180
1892
2443
2418
1241
1862
1721
933
665
Regression
2029
2801
809
1606
1193
1208
1119
1950
2408
2388
1227
1882
1714
972
598
Deviation
8.7
-62.0
38.9
-16.6
119.8
3.6
44.0
-97.4
-16.6
-21.7
-6.5
-57.0
-26.7
-52.0
64.2
5/9
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
2404
1151
657
2454
394
1242
1707
2596
1525
1697
475
1495
949
606
1151
1172
1576
586
1505
1465
2212
1434
333
2222
1121
586
2414
404
1273
1737
2687
1545
1656
535
1525
899
566
1162
1202
1707
596
1485
1414
2343
1374
343
2428
1165
600
2438
509
1246
1620
2690
1468
1711
398
1256
802
680
1125
1246
1660
660
1347
1367
2185
1286
317
2306
1165
600
2357
468
1226
1539
2529
1418
1620
398
1438
923
509
1054
1155
1650
529
1296
1549
2165
1347
377
2313
1136
621
2434
399
1257
1722
2641
1535
1677
505
1510
924
586
1156
1187
1641
591
1495
1439
2278
1404
338
2367
1165
600
2397
488
1236
1579
2609
1443
1665
398
1347
862
594
1089
1200
1655
594
1322
1458
2175
1317
347
2265
1119
617
2383
401
1237
1690
2585
1508
1646
504
1483
913
583
1139
1168
1611
588
1468
1414
2231
1380
342
54.1
28.8
-21.7
-36.8
89.5
-21.6
-142.9
-31.8
-92.3
-11.6
-107.5
-163.1
-62.1
8.7
-67.1
13.7
13.7
3.6
-173.2
18.8
-102.5
-87.3
8.6
U R E A liq u ic o lo r U rin e
3000
2500
Test mg/dl
2000
1500
1000
500
0
0
500
1000
1500
2000
2500
3000
R e fe re n c e m g / d l
Conclusion:
Both methods showed a good agreement and no significant deviation could be observed with
any specific sample.
6 Interferences
Interference by triglycerides, hemoglobin, bilirubin and ascorbic acid at 578 nm have been studied by adding
known amounts of the potentially interfering substance to a known sample.
Recoveries have been analyzed according to the method of Glick et al. (Clin.Chem. 1986, 32 470-5).
The results are summarised in the following table.
Triglyc.
Analyt.
Dev.
Hemogl
Result
Analyt.
Dev.
Bilirubi
Result
Analyt.
Dev.
Result
Ascob.
Analyt.
acid
Result
Dev.
mg/dl
mg/dl
%
mg/dl
mg/dl
%
mg/ml
mg/dl
%
mg/ml
mg/dl
%
0
55.60
100
0
64.20
100
0
64.80
100
0
66.95
100
250
58.65
105.5
50
64.45
100.4
4
64.95
100.2
2
66.80
99.8
500
61.45
110.5
100
63.85
99.5
8
64.90
100.2
4
66.95
100.0
750
62.75
112.9
150
63.70
99.2
12
64.95
100.2
6
66.75
99.7
1000
64.00
115.1
200
64.75
100.9
16
63.80
98.5
8
67.10
100.2
1250
66.80
120.1
250
64.05
99.8
20
64.40
99.4
10
67.25
100.4
1500
69.05
124.2
300
64.00
99.7
24
64.65
99.8
12
67.40
100.7
1750
70.70
127.2
350
63.45
98.8
28
64.20
99.1
14
66.60
99.5
2000
72.65
130.7
400
63.00
98.1
32
64.85
100.1
16
66.55
99.4
Design Verification and Product Data for UREA LIQUICOLOR
Rev. 009
6/9
2250
73.70
132.6
450
64.75
100.9
36
64.30
99.2
18
67.80
101.3
2500
76.20
137.1
500
64.50
100.5
40
64.35
99.3
20
66.95
100.0
Glick
3
1
1
1
Conclusion: No significant interfering effect has been seen up to following concentrations:
Hemoglobin 500 mg/dl
Bilirubin
40 mg/dl
Ascorbate
20 mg/dl.
Triglycerides showed a positive interfering effect of about 10% at 500 mg/dl and about 15% at 1000
mg/dl triglycerides.
Furthermore, interferences of haemoglobin and bilirubin at 546 nm measurement wavelength have been studied
by adding known amounts of the potentially interfering substance to a known sample. Recoveries have been
analyzed according to the method of Glick et al. (Clin.Chem. 1986, 32 470-5).
Hemogl
Analyt.
Dev.
Bilirubi
Result mg/dl
mg/dl
Analyt.
Dev.
Result mg/dl
%
mg/ml
0
59.40
100
0
61.10
100
%
50
55.25
93.0
4
60.60
99.2
100
52.85
89.0
8
60.90
99.7
150
50.70
85.4
12
59.75
97.8
200
49.35
83.1
16
59.35
97.1
250
48.55
81.7
20
59.85
98.0
300
46.15
77.7
24
59.55
97.5
350
45.25
76.2
28
60.35
98.8
400
45.05
75.8
32
61.10
100.0
450
44.10
74.2
36
60.45
98.9
500
43.25
72.8
40
60.15
98.4
Glick
3
1
Conclusion: No significant interfering effect has been seen up to following concentrations:
Bilirubin
40 mg/dl
Hemoglobin interferes significantly (>10% deviation) above 50 mg/dl.
Design Verification and Product Data for UREA LIQUICOLOR
Rev. 009
7/9
7 Stability
Recovery in Control Sera
For real-time study, one of the available multipurpose reagent kit sizes was used. The UREA liquicolor multipurpose
reagent was stored at 2…8°C. The real-time stability was tested up to 125% of the shelf life at several intervals.
CONTROL SERUM RECOVERY
Control
LOT
serum
Reference
23 months
24 months
26 months
Target
Range
Result
Deviation
Result
Deviation
Result
Deviation
Result
Deviation
mg/dl
mg/dl
mg/dl
(%)
mg/dl
(%)
mg/dl
(%)
mg/dl
(%)
HumaTrol N
# 018
51.2
38.9 – 63.5
40.8
1.25
41.5
1.23
41.7
1.22
41.1
1.24
HumaTrol P
# 016
172
131 - 213
150.8
1.13
151.6
1.13
41.6
1.22
150.8
1.14
SERODOS
# 6868
52
39.5 – 64.5
50.6
1.02
51.7
1.00
50.8
1.02
50.6
1.02
SERO.Plus
# 6795
173
131 - 214
151.2
1.13
154.4
1.12
153.3
1.12
151.3
1.14
Precinorm
#159296
42.2
32.0 – 52.4
44.8
0.95
44.0
0.96
43.9
0.96
43.4
0.97
Precipath
#165215
146
110 - 182
146
110 - 182
147.3
0.99
146.9
0.99
144.4
1.00
Mean
106
97.3
98.4
97.7
96.9
Conclusion: The control recovery data for UREA liquicolor multipurpose reagent are indicative for a real-time
stability of 24 months at 2…8°C.
Linearity
Four independent lots have been stored under the recommended conditions for up to 26 months 2…8°C. A high
concentration pool serum was employed and dilutions with phys. saline were made. The analysed concentrations
were calculated vs. the regression line. Deviation from the regression line are expressed in absolute and relative
values.
Reference
20 months
24 months
26 months
LOT H059
R1 LOT CD321
R1 LOT UJ 074
LOT H011
R2 LOT CD622
R2 LOT UJ060
Enz. LOT CD620A
Enz. LOT UJ061
High pool
Analytical Data
Regression
Analytical Data
Regression
Analytical Data
Regression
Analytical Data
Regression
Content (%)
mg/dl
Line (%)
mg/dl
Line (%)
mg/dl
Line (%)
mg/dl
Line (%)
0
-0.70
-137.23
1.15
-373.81
0.45
-314.29
1.00
-166.67
10
54.20
3.95
42.20
-2.62
44.95
3.21
52.50
14.01
20
104
1.56
87.09
-0.85
84.65
-3.05
76.30
-18.48
30
154
0.62
131
-1.07
130
-1.16
146
3.72
40
201
-1.00
175
0.97
177
1.21
192
1.64
50
253
-0.11
218
0.64
221
1.15
241
2.14
60
301
-0.64
262
0.42
265
1.09
293
3.17
70
347
-1.98
306
0.83
311
1.48
347
4.63
80
387
-4.15
350
1.08
351
0.47
388
2.48
90
421
-7.23
393
2.59
401
1.83
434
1.70
100
460
-8.90
437
-0.04
435
-0.44
472
-0.42
Temperature Stress Results
In addition to the real-time storage, temperature stress tests have been applied on the UREA liquicolor test.
Stressed reagents have been compared with fresh ones (see chapter 4).
Conclusion:
The results obtained with reagents stored for even 26 months under real-time conditions and
with stressed reagents. From the above linearity comparison the stability claim of 24 months
2…8°C from the date of manufacture has been confirmed.
Design Verification and Product Data for UREA LIQUICOLOR
Rev. 009
8/9
8 Stability Working Reagent
Recovery in Control Sera
The working reagent has been stored 3 weeks at 25°C and 4 weeks at 2…8°C. Commercially available control sera
have been employed. The control sera have been reconstituted/prepared according to the manufacturer’s
instructions. Twofold determinations of each control serum have been performed with the UREA liquicolor test
method. The means of the twofold determinations have been calculated and compared with the target values.
CONTROL SERUM RECOVERY
Control
LOT
serum
3 weeks 25°C
4 weeks 2…8°C
Target
Range
Result
Deviation
Result
Deviation
mg/dl
mg/dl
mg/dl
(%)
mg/dl
(%)
HumaTrol N
# 016
51.1
38.9-63.5
45.9
-10.22
49.2
-3.76
HumaTrol P
# 018
172
131-213
165.7
-3.65
165.7
-3.69
SERODOS
# 6868
52
39.5-64.5
58.4
12.38
53.7
3.27
SERODOS plus
# 6795
172.7
131.3-214.1
171.5
-0.72
160.8
-6.88
Precinorm
#154916
43.2
32.7-53.7
45.5
5.32
44.1
1.99
Precipath
#199459
140
107-173
154.3
10.20
150.5
7.47
641.28
13.32
623.9
-1.59
Summary
1074
Linearity
The working reagent has been stored 3 weeks at 25°C and 4 weeks at 2…8°C. A high concentration pool serum was
employed and dilutions with phys. saline were made. The analysed concentrations were calculated vs. the
regression line. Deviation from the regression line are expressed in absolute and relative values.
Reference
3 weeks 25°C
4 weeks 2…8°C
High pool
Analytical Data
Regression Line
Analytical Data
Regression Line
Analytical Data
Content (%)
mg/dl
(%)
mg/dl
(%)
mg/dl
(%)
0
1.70
1077
0.00
-100
0.00
-100
10
43.7
2.38
41.6
0.37
40.5
-1.38
20
84.7
-0.62
86.3
-2.45
84.5
-0.54
30
124
-2.83
132
-2.50
128
-0.58
40
168
-1.12
178
-2.27
170
-1.64
50
216
1.27
229
-0.33
217
0.29
60
254
-0.74
278
0.40
260
-0.31
70
303
1.70
325
0.29
303
-0.49
80
338
-0.70
373
0.72
352
0.98
90
380
-0.84
424
1.62
393
0.27
100
420
-1.31
461
-0.74
430
-1.45
Conclusion:
Regression Line
The above results clearly demonstrate the stability of the working reagent for 2 weeks at 25°C
and for 4 weeks at 2…8°C.
9 Traceability
The UREA liquicolor test has to be calibrated with a standard. UREA concentration in the kit standard has been
verified with the reference material NIST SRM 909c. UREA liquicolor revealed recovery well within the
reproducibility limits of the method.
Conclusion:
The method is traceable to the reference preparation NIST SRM 909c.
Design Verification and Product Data for UREA LIQUICOLOR
Rev. 009
9/9