Design Verification

Cholesterol liquicolor

Contents

1 Introduction .................................................................................................................................................................. 2
2 Imprecision .................................................................................................................................................................... 2
3 Linearity and Detection Limit ...................................................................................................................................... 2
3.1 Linearity ....................................................................................................................................................................................... 2
3.2 Detection Limit ......................................................................................................................................................................... 3
4 Recovery in Control Sera .............................................................................................................................................. 3
5 Comparison of Methods ............................................................................................................................................... 4
6. Stability Data ................................................................................................................................................................ 5
6.1 Stability by Accelerated Stress Tests ................................................................................................................................ 5
6.2 Recovery of Control Sera ....................................................................................................................................................... 6
6.3 Real Time Stability .................................................................................................................................................................. 6
6.4 Open Vial Stability ................................................................................................................................................................... 7
6 Interference ................................................................................................................................................................... 8
7 Traceability .................................................................................................................................................................... 9

Form: 4.2-01.06-04 1/9

Rev. 009 | valid of 24.11.2020
1 Introduction
The performance characteristics of the CHOLESTEROL liquicolor have been tested and documented in order to
verify the clinical usefulness and compliance with the essential requirements of directive 98/79/EC.

2 Imprecision
The imprecision (within-run and day-to-day) of CHOLESTEROL liquicolor was calculated from six determinations on
ten consecutive days. Commercial control sera with low, medium and high cholesterol levels were employed as
sample material. The tests were performed on AU 400 system. The mean, standard deviation (SD) and coefficient of
variation (%CV) were calculated.

Mean analyte concentration Intra-assay Inter-assay

(mg/dl) SD (mg/dl) %CV SD (mg/dl) %CV
216 1.4 0.7 2.0 0.9
292 1.8 0.6 2.0 0.7
638 4.0 0.6 5.2 0.8

Conclusion: The reagent shows a good intra- and inter-assay precision in the low, middle, and high
concentration range.

3 Linearity and Detection Limit

3.1 Linearity
The linearity of the CHOLESTEROL liquicolor method was controlled by employing dilutions of a high concentration
pool serum with physiological saline. The analysed concentrations were calculated vs. the regression line.
Deviations from the regression line are expressed in absolute and relative values.
As an example data measured on the Hitachi 717 are reported.

High Pool Analytical Data Regressed Data Deviation from Regression Line
Content (%) (mg/dl) (mg/dl) (mg/dl) (%)
0 -0.65 -1.50 0.85 -56.7
10 91,3 91.0 0.34 0.38
20 179 183 -4.66 -2.54
30 277 276 1.18 0.43
40 367 368 -1.33 -0.36
50 472 461 11.0 2.38
60 546 553 -7.34 -1.33
70 657 646 11.2 1.74
80 744 738 6.24 0.85
90 833 831 2.14 0.26
100 920 923 -3.12 -0.34

Design Verification and Product Data for CHOLESTEROL liquicolor 2/9

Rev. 009
 LINEARITY
1000

Linear Regression
900
Error Bars = 2SD

800

MEASURED ANALYTE CONCENTRATION (m g/dl)

700

600

500

400

300

200

100

0
0 20 40 60 80 100
HIGH POOL CONTENT (%)

Conclusion: CHOLESTEROL liquicolor is linear up to 750 mg/dl.

3.2 Detection Limit

A 20-fold determination of a ‘0’ sample (phys. saline) on a HS100 analyser revealed an absolute mean of -3.93
mg/dl and a SD of 0.98mg/dl. From the three-fold standard deviation the detection limit can therefore be
calculated on the base of mean +3 SD to 6.87mg/dl.

4 Recovery in Control Sera

Commercially available control sera have been employed. The control sera have been reconstituted/prepared
according to the manufacturer’s instructions. Fivefold determinations of each control serum have been performed
with CHOLESTEROL liquicolor and a commercially available cholesterol test as reference. The mean and the
coefficient of variation of the twofold determinations have been calculated and the means compared with the
target value of the respective control serum. The results are summarised below.

Reference CHOLESTEROL liquicolor

Control Lot Target Range Result Deviation Result Deviation
Serum mg/dl mg/dl mg/dl (%) mg/dl (%)
HumaTrol N # 018 158 129 – 186 157 -0.63 159 0.63
HumaTrol P # 016 238 193 – 282 239 0.42 243 2.10
SERODOS # 6868 172.6 141.5–203.7 171 -0.58 176 1.97
SERODOS plus # 6795 263.8 216 – 311 246 -6.75 264 0.08
Precinorm # 154916 107 89 – 125 107 0.00 114 6.54
Precipath # 199459 184 151 - 217 183 -0.54 188 2.17
Sum 1123.4 1103 1144
Mean 187.23 183.83 -1.35 190.67 2.24

Conclusion: All control sera were recovered within the specified ranges. There was a good agreement
observed of the reference test and CHOLESTEROL liquicolor in regard to the sum of recoveries
and mean deviations.

Design Verification and Product Data for CHOLESTEROL liquicolor 3/9

Rev. 009
5 Comparison of Methods
CHOLESTEROL liquicolor lot # 0117 on AU 400 analyzer has been compared against Beckman Coulter Cholesterol
reagent lot # 5076 on AU 400 analyzer. Control sera as well as patient samples have been employed in the
comparison (N = 54). The results have been evaluated by a non-parametric regression analysis according to Passing
& Bablok. Acceptance criteria were r ≥ 0.95 and a slope between 0.95 and 1.05. The linear regression obtained could
be described as follows:
Y= 1.004 X + 2.48
r= 0.9985
XMean = 210 mg/dl
YMen = 213 mg/dl

Sample Mean Reference [mg/dl] Mean Test [mg/dl]

No. Cholesterol Beckman Coulter CHOLESTEROL liquicolor
1 227 230
2 222 226
3 246 251
4 300 300
5 175 178
6 244 245
7 240 243
8 215 218
9 246 250
10 294 294
11 248 253
12 197 201
13 171 176
14 135 138
15 180 181
16 119 123
17 170 172
18 160 163
19 160 164
20 192 194
21 166 167
22 153 154
23 147 150
24 228 232
25 216 220
26 245 245
27 122 126
28 117 123
29 253 257
30 176 180
31 189 195
32 198 198
33 183 187
34 144 146
35 249 253
36 202 204
37 236 238
38 222 224
39 244 247
40 160 164
41 147 149
42 161 165

Design Verification and Product Data for CHOLESTEROL liquicolor 4/9

Rev. 009
 43 217 220
44 214 220
45 253 258
46 308 318
47 292 302
48 234 240
49 289 295
50 274 264
51 267 270
52 291 295
53 191 194
54 216 218

Conclusion: The method showed a good agreement and no significant deviation could be observed.

6. Stability Data

6.1 Stability by Accelerated Stress Tests

Linearity
The linearity of the CHOLESTEROL liquicolor lot # 0117 calibrated with Autocal lot # H014 was controlled according
to the procedure already described in 3. The reagents were stressed 14 days at 37°C. Acceptance criteria is relative
deviation of <10%.
As an example data measured on the AU 400 system are reported.

High Pool Analytical data Regressed data Deviation from Regression line
Content (%) mg/dl mg/dl mg/dl (%)
0 0 0
10 122 116 5.93 5.1
20 242 232 9.77 4.2
30 365 348 17.2 4.9
40 484 464 19.6 4.2
50 606 580 26.3 4.5
60 719 696 22.6 3.2
70 838 812 26.2 3.2
80 943 928 14.8 1.6
90 1050 1044 6.3 0.6
100 1075 1160 -85.2 -7.3

Design Verification and Product Data for CHOLESTEROL liquicolor 5/9

Rev. 009
Conclusion: These data indicate that CHOLESTEROL liquicolor retains its linearity of up to 750 mg/dl over the
claimed stability time-frame.

6.2 Recovery of Control Sera

Commericaly available control sera have been employed. The control sera have been reconstituted/prepared
according to the manufacturer’s instructions. Threefold determinations of each control serum have been
performed with CHOLESTEROL liquid reagent lot # 0117. The mean values obtained with fresh and stressed (14
days at 37°C) reagents have been calculated and compared with the target values of the respective control sera and
means established with fresh reagent. Acceptance criteria are deviation from mean of fresh reagent <10% and
findings within control ranges.
As an example data measured on the AU 400 system are reported.

CONTROL SERUM RECOVERY Fresh Cholesterol reagent lot # 0117

stressed 14 days at 37°C
Control Lot Target Range Result Result Deviation
mg/dl mg/dl mg/dl mg/dl (%)
Humatrol N #0002 161 138 - 183 163 163 -0,2
Humatrol P #0002 246 212 - 280 254 256 0,7
SERODOS #0002 158 136 - 180 166 166 -0,2
SERODOS Plus #0002 239 206 - 272 243 245 0,6
Precinorm #168004 90,5 77,0-104,0 94 93 -1,0
Precipath #167102 196 166 - 226 207 207 0,0
Olympus 1 #0033 157 135-179 164 164 -0,1
Olympus 2 #0034 277 239-316 291 293 0,6

Sum 1525 1584 1587

Mean 191 198 198 0.2

Conclusion: Accelerated stress data are indicative for a real-time stability of 24 months at a storage
temperature of 2...8°C, confirming the shelf life of 20 months.

6.3 Real Time Stability

The stability of CHOLESTEROL liquicolor has been tested on real-time storage conditions over a period of 19, 20, 21
and 23 months for three independent production lots (H014, exp. date: May 2003; H012, exp. date: August 2003;
H011, exp. date: July 2003; H010, exp. date: May 2003), measurements were made on Hitachi 717. Acceptance
criteria were based on the recovery of controls and linearity.

Design Verification and Product Data for CHOLESTEROL liquicolor 6/9

Rev. 009
 Lot # H014 Lot # H012 Lot # H011 Lot # H010
exp. date 9/2003 exp. date 8/2003 exp. date 7/2003 exp. date 5/2003
19 months 20 months 21 months 23 months
Control Target Range fresh 10/2003 fresh 10/2003 fresh 10/2003 fresh 10/2003
mg/dl mg/dl mg/dl mg/dl mg/dl mg/dl mg/dl mg/dl mg/dl mg/dl
Humatrol N 166 136- 167 162 154 162 165 162
lot# 019 196 167 163 152 163 164 163
Humatrol P 254 208- 249 256 256 256 256
# 017 299 248 248 248 248
Serodos 173 142- 172 172 162 173 183 173 181 173
lot# 6868 204 180 172 164 172 179 172 178 172
Serodos plus 256 223- 255 255 255 255
lot# 6796 289 254 254 254 254
Precinorm 98.1 80.4- 104 104 104 104
Lot# 159296 116 103 103 103 103
Precipath 188 155- 190 190 190 190
Lot# 151348 221 190 190 190 190

Conclusion: All real time stability data for control recovery confirm the shelf life of 20 months for
CHOLESTEROL liquicolor Multipurpose reagent.

6.4 Open Vial Stability

Open vial stability for CHOLESTEROL liquicolor lot # 0117 was controlled according to the procedure already
described in 3. The reagents in opened vials were kept on-board for 4 weeks. Acceptance criteria is relative
deviation of <10%.
As an example data measured on the AU 400 system are reported.

High Pool Analytical data Regressed data Deviation from Regression line
Content (%) mg/dl mg/dl mg/dl (%)
0 -0.4 0 -0.37
10 123 116 6.56 5.7
20 243 232 10.6 4.6
30 360 348 11.7 3.4
40 478 464 13.8 3.0
50 589 580 9.13 1.6
60 703 696 6.91 1.0
70 823 812 10.5 1.3
80 934 928 6.19 0.7
90 1047 1044 3.03 0.3
100 1142 1160 -18.0 -1.6

Design Verification and Product Data for CHOLESTEROL liquicolor 7/9

Rev. 009
Conclusion: These data confirm CHOLESTEROL liquicolor linearity of up to 750 mg/dl over the claimed
stability time-frame.

Recovery of control sera

Commericaly available control sera have been employed. The control sera have been reconstituted/prepared
according to the manufacturer’s instructions. Threefold determinations of each control serum have been
performed with CHOLESTEROL liquid reagent lot # 0117. The mean values obtained with fresh and opend (28 days
opened) reagents have been calculated and compared with the target values of the respective control sera and
means established with fresh reagent. Acceptance criteria are deviation from mean of fresh reagent <10% and
findings within control ranges.

CONTROL SERUM RECOVERY CHOLESTEROL liquicolor lot # 0117 opened 28 days

Fresh Calibration stability Open vial stabilty
Control Serum Lot Target Range Result Result Deviation Result Deviation
mg/dl mg/dl mg/dl mg/dl (%) mg/dl (%)
HumaTrol N # 0002 161 138 – 183 162 162 0,0 162 0,0
HumaTrol P # 0002 246 212 – 280 251 247 -1,5 247 -1,5
SERODOS # 0002 158 136 – 180 163 159 -2,2 159 -2,2
SERODOS plus # 0002 239 206 – 272 243 237 -2,4 237 -2,4
SERODOS plus #0003 264 227 - 301 265 263 -1,0 263 -1,0
Precinorm #168004 90.5 77.0 – 104 93 92 -1,7 92 -1,7
Precipath #167102 196 166 – 226 204 201 -1,2 201 -1,2
Olympus 1 #0033 157 135 – 179 160 158 -1,3 158 -1,3
Olympus 2 #0034 277 239 - 316 283 280 -0,8 280 -0,8

Sum 895 1823 1799 1799

Mean 179 203 200 200

Conclusion: The open vial stability and calibration stability is confirmed for a minimum of 28 days.

6 Interference
Interferences by lipemia (intralipid), haemoglobin, bilirubin and ascorbic acid have been studied by adding known
amounts of the potentially interfering substances to a known sample. Recoveries have been analyzed according to
the method of Glick et al. (Clin.Chem. 1986, 32 470-5).

Intralipid Result Recovery Hemo. Result Recovery Bilirubin Result Recovery Ascorb. Result Recovery
mg/dl mg/dl % mg/dl mg/dl % mg/dl mg/dl % Acid. mg/dl %
mg/dl
0 183 100.0 0 179 100.0 0 181 100.0 0 183 100.0
100 182 99.5 50 182 102.2 4 178 98.2 2 175 95.6
200 185 101.2 100 182 101.8 8 173 95.5 4 175 95.7
300 183 100.0 150 184 103.2 12 170 93.6 6 169 92.1
400 185 101.1 200 183 102.8 16 164 90.6 8 167 91.1
500 187 102.5 250 187 104.8 20 166 91.7 10 160 87.2
600 187 102.4 300 188 105.5 24 165 91.2 12 158 86.4
700 188 102.9 350 189 106.1 28 167 92.0 14 156 84.9
800 182 99.8 400 194 108.8 32 168 92.7 16 150 82.0
900 185 101.5 450 192 107.3 36 170 93.9 18 147 80.2
1000 187 102.1 500 194 108.9 40 177 97.5 20 142 77.2
Glick 1 2 2 3

Design Verification and Product Data for CHOLESTEROL liquicolor 8/9

Rev. 009
Conclusion: Through the added Lipid Clearing Factor (LCF), lipemic samples could be used. Hemoglobin up to
500 mg/dl and bilirubin up to 40 mg/dl do not significantly interfere with the test. Falsely low
results have been obtained in samples containing ascorbic acid concentrations above 8 mg/dl.

7 Traceability
The cholesterol concentration in the standard supplied with CHOLESTEROL liquicolor has been calibrated against
the reference material NIST SRM 909c. When the reference material was applied as sample, the recovery was found
well within the reproducibility limits of the method. The kit standard and AUTOCAL are traceable to NIST SRM 909c.

Design Verification and Product Data for CHOLESTEROL liquicolor 9/9

Rev. 009