Design Verification

opt. liquicolor

1 Introduction .................................................................................................................................................................. 2
2 Imprecision .................................................................................................................................................................... 2
3 Linearity and Sensitivity............................................................................................................................................... 2
3.1 Linearity ....................................................................................................................................................................................... 2
3.1 Sensitivity.................................................................................................................................................................................... 3
4 Comparison of Methods ............................................................................................................................................... 3
5 Stability.......................................................................................................................................................................... 4
5.1 Stability Working Reagent .................................................................................................................................................... 4
Recovery of Control Sera ......................................................................................................................................................................... 4
Linearity......................................................................................................................................................................................................... 5
5.2 Real Time Stability ................................................................................................................................................................... 5
6 Interference ................................................................................................................................................................... 8
7 Traceability .................................................................................................................................................................... 9

Form: 4.2-01.06-04 1/9

Alkaline Phosphatase opt. liquicolor | Rev. 007 | valid of 01.02.2019
1 Introduction
The performance characteristics of the ALKALINE PHOSPHATASE opt. liquicolor opt. 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 ALKALINE PHOSPHATASE opt. liquicolor was calculated from six
determinations on ten consecutive days. Commercial control sera with low, medium and high alkaline
Phosphatase levels were employed as sample material.

Hitachi 717

Analyte concentration Intra-assay Inter-assay

(U/l) SD (U/l) %CV SD (U/l) %CV
42 1.26 3.0 1.5 3.5
267 7.27 2.7 8.3 3.1
938 22.2 2.4 25 2.7

3 Linearity and Sensitivity

3.1 Linearity
The linearity of ALKALINE PHOSPHATASE opt. liquicolor was controlled by employing a high concentration pool
serum and dilutions with phys. saline. The analysed concentrations were calculated vs. the regression line.
Deviation 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 (%) (U/l) (U/l) (U/l) (%)
0 -1.05 -11.6 10.5 -90.9
10 201 194 7.23 3.72
20 391 400 -9.12 -2.28
30 606 606 0.74 0.12
40 799 812 -12.1 -1.50
50 998 1017 -18.9 -1.86
60 1226 1223 3.11 0.25
70 1432 1429 3.21 0.22
80 1646 1635 11.3 0.69
90 1861 1840 20.3 1.10
100 2030 2046 -16.2 -0.79

Design Verification and Product Data for Alkaline Phosphatase opt. liquicolor 2/9
Rev. 007
Conclusion: The ALKALINE PHOSPHATASE opt. liquicolor is linear up to about 2000 U/l..

3.1 Sensitivity
A 20-fold determination of a 0 calibrator (phys. saline) on a Hitachi 717 analyser revealed an absolute mean of
0.87 U/l and a SD of 1.16 U/l. Sensitivity was calculated on base of mean + 3SD = 3.48 U/l.

4 Comparison of Methods
ALKALINE PHOSPHATASE opt. liquicolor has been compared with a commercially available alkaline Phosphatase
method. Control sera as well as patient samples have been employed in the comparison (N=54).
The results have been evaluated by a main component analysis. The linear regression obtained could be described
as follows:
ALKALINE PHOSPHATASE opt. liquicolor (Y) = 0.995 * Ref. method (X) - 3.506
The coefficient of correlation was determined to r = 0.996.
Evaluation of the method comparison by a non-parametric model according to Bablok&Passing yielded the
following results:
Reference (REF)
Lowest value = 106.0000
Highest value = 331.5000
Arithmetic mean = 188.1111
Median = 178.9500

Design Verification and Product Data for Alkaline Phosphatase opt. liquicolor 3/9
Rev. 007
Standard deviation = 57.3444
Standard error of the mean = 7.8036

HUMAN AP_DGKCH
Lowest value = 104.8000
Highest value = 326.7000
Arithmetic mean = 183.6981
Median = 174.7500
Standard deviation = 57.3240
Standard error of the mean = 7.8008

Y = -1,8810 + 0,9881 X
Intercept A :-1.8810;95% CI : -6.6865 to 3.2489
Slope B : 0.9881;95% CI : 0.9572 to 1.0148

Cusum test for linearity: No significant deviation from linearity (P>0.10)

Method Comparison Alkaline Phosphatase

DGKCh
350

300

AP 250
_D
G
K
C
H,
200
U/l

150

100
100 150 200 250 300 350
REF, U/l

Conclusion: The comparison study confirmed an excellent agreement between both methods.

5 Stability
5.1 Stability Working Reagent

Recovery of 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 ALKALINE PHOSPHATASE opt. liquicolor. The means of the fivefold determinations have been calculated and
compared with the values of fresh working reagent.

Design Verification and Product Data for Alkaline Phosphatase opt. liquicolor 4/9
Rev. 007
 Control serum recovery Working Reagent fresh Working Reagent Working Reagent
5 days 25°C 7 days 25°C
Control LOT Result Result Deviation Result Deviation
Serum U/l U/l (%) U/l (%)
HumaTrol N # 018 203 210 -3.45 197 -3.06
HumaTrol P # 016 513 516 -0.58 498 -3.03
SERODOS # 6868 180 184 -2.23 181 0.55
SERODOS plus # 6794 418 386 7.66 391 -6.5
SERODOS plus # 6795 411 423 2.92 430 4.63
Mean 345 343.8 3.37 339.4 1.48

Linearity
The linearity of the ALKALINE PHOSPHATASE opt. liquicolor was controlled according to the procedure already
described in 2.1. The working reagents were stressed 5 and 7 days at 25°C.

ALKALINE PHOSPHATASE opt. liquicolor ALKALINE PHOSPHATASE opt. liquicolor

working reagent 5 days 25°C working reagent 7 days 25°C
High Pool Analytical Regressed Deviation from Regression Analytical Regressed Deviation from Regression
Data Data Line Data Data Line
Content (%) U/l U/l U/l (%) U/l U/l U/l (%)
0 1.15 14.51 -13.36 -92.07 -0.35 17.63 -17.98 -101.99
10 236 234 2.40 1.03 224 232 -7.37 -3.18
20 465 453 12.72 2.81 455 446 9.50 2.13
30 665 672 -6.62 -0.99 691 660 31.31 4.74
40 894 891 2.64 0.30 894 874 19.68 2.25
50 1122 1110 11.51 1.04 1062 1088 -25.81 -2.37
60 1340 1329 11.02 0.83 1292 1302 -10.10 -0.78
70 1528 1548 -20.32 -1.31 1517 1517 0.77 0.05
80 1690 1768 -77.10 -4.36 1717 1731 -13.82 -0.80
90 1859 1987 -128 -6.44 1894 1945 -51.01 -2.62
100 2045 2206 -161 -7.30 2011 2159 -148.14 -6.86

Conclusion: The working reagent is stable for 4 weeks at 4°C and for 5 days at 15…25°C.

5.2 Real Time Stability

For real-time study, one of the available multipurpose reagent kit sizes was used. The ALKALINE PHOSPHATASE opt.
liquicolor was stored at 2…8°C. The real-time stability was tested up to 125% of the shelf life at several intervals.

5.2.1 Recovery of Control Sera

A number of commercially available control sera have been employed according to the procedure already described
in section 4 on AU 400 & AU 480. The mean values (n=2) obtained with fresh reagent (=reference) and 3 different
reagent LOTs have been calculated and compared.
Criteria
Check Acceptance criteria
Recovery within range
Deviation result mean from fresh mean ≤ 10%

Used Material
Reagent Manufacturer REF LOT
ALKALINE PHOSPHATASE opt. liquicolor HUMAN 12037 16001; 17003/reference
ALKALINE PHOSPHATASE opt. liquicolor HUMAN 12037 15002 /25 months
ALKALINE PHOSPHATASE opt. liquicolor HUMAN 12037 15004 /25 months
ALKALINE PHOSPHATASE opt. liquicolor HUMAN 12017 16002/25 months
AUTOCAL HUMAN 13160 H015; H016
Sample HUMAN’s and commercial controls (Beckman Coulter)

Design Verification and Product Data for Alkaline Phosphatase opt. liquicolor 5/9
Rev. 007
Results
Control recovery ALKALINE PHOSPHATASE opt. liquicolor,
REF 12037
Reference Test
LOT 16001 LOT 15002
Fresh 22.5 months
Name LOT Target Range Result Result Within range
U/l U/l U/l U/l Y/N
HumaTrol N 0004 202 152 – 253 195 198 Yes
HumaTrol P 0003 952 714 – 1190 898 874 Yes
SERODOS 0003 333 250 – 416 317 314 Yes
SERODOS
plus 0003 595 446 – 744 630 619 Yes
Control serum L1 0037 171 128 – 214 186 202 Yes
Control serum L1 0038 689 517 - 861 673 733 Yes
Dev.%
Mean 490 483 490 0.1

Control recovery ALKALINE PHOSPHATASE opt. liquicolor,

REF 12037
Reference Test
LOT 16001 LOT 15004
Fresh 25 months
Name LOT Target Range Result Result Within range
U/l U/l U/l U/l Y/N
HumaTrol N 0005 187 140 - 234 181 180 Yes
HumaTrol P 0003 952 714 - 1190 889 878 Yes
SERODOS 0004 257 193 - 321 280 276 Yes
plus
SERODOS 0004 513 385 - 641 491 490 Yes
Control serum L1 0037 171 128 - 214 219* 219* No
Control serum L1 0038 689 517 - 861 782 770 Yes
Dev.%
Mean 462 474 469 -1.0
*out of range; not excluded; out of control range is uncritical for this study.

Control recovery ALKALINE PHOSPHATASE opt. liquicolor,

REF 12017
Reference Test
LOT 17003 LOT 16002
Fresh 25 months
Name LOT Target Range Result Result Within range
U/l U/l U/l U/l Y/N
HumaTrol N 0005 187 140 - 234 164 180 Yes
HumaTrol P 0004 764 573 - 955 700 725 Yes
SERODOS 0004 257 193 - 321 251 253 Yes
plus
SERODOS 0004 513 385 - 641 472 483 Yes
Control serum L1 0037 171 128 - 214 207 200 Yes
Control serum L1 0038 689 517 - 861 723 717 Yes
Dev.%
Mean 430 420 426 2.0

Control recovery for ALKALINE PHOSPHATASE opt. liquicolor Multipurpose reagent are indicative for a real-time
stability of 18 months.

Design Verification and Product Data for Alkaline Phosphatase opt. liquicolor 6/9
Rev. 007
 5.2.2 Linearity
The linearity of ALKALINE PHOSPHATASE opt. liquicolor Multipurpose reagent was controlled according to the
procedure already described in section 3 using high concentrated pool serum on an AU 480. The linearity was
controlled for 3 different reagent LOTs.

Criteria
Linearity Acceptance
criteria
Deviation from regression line ≤ 10%

Used Material
Reagent Manufacturer REF LOT
ALKALINE PHOSPHATASE opt. liquicolor HUMAN 12037 15002/20 months
ALKALINE PHOSPHATASE opt. liquicolor HUMAN 12017 15004/25 months
ALKALINE PHOSPHATASE opt. liquicolor HUMAN 12017 16002/25 months
Sample Diluted pool sera, LOT CEN102A; CEN131; BOG190

Results
ALKALINE PHOSPHATASE opt. liquicolor, REF 12037, LOT 15002/20 months
High Pool Content Analytical Data Calculated Data Deviation from Regression Line
(%) (U/l) (U/l) (U/l) (%)
0 -0.31 0.00 -0.31
10 231 250 -19 -7.5
20 544 500 44 8.8
30 714 750 -36 -4.8
40 985 1000 -15 -1.5
50 1260 1250 10 0.8
60 1511 1500 11 0.8
70 1789 1750 39 2.2
80 2067 2000 67 3.3
90 2246 2250 -4 -0.2
100 2495 2500 -5 -0.2

ALKALINE PHOSPHATASE opt. liquicolor, REF 12017, LOT 15004/25 months

High Pool Content Analytical Data Calculated Data Deviation from Regression Line
(%) (U/l) (U/l) (U/l) (%)
0 -0.07 0 -0.07
10 290 281 9 3.3
20 569 562 7 1.3
30 842 842 0 0.0
40 1140 1123 17 1.5
50 1391 1404 -13 -0.9
60 1669 1685 -16 -0.9
70 1946 1966 -20 -1.0
80 2241 2246 -6 -0.2
90 2465 2527 -62 -2.4
100 2889 2808 81 2.9

ALKALINE PHOSPHATASE opt. liquicolor, REF REAGENTNO, LOT X1/XX months

High Pool Content Analytical Data Calculated Data Deviation from Regression Line
(%) (U/l) (U/l) (U/l) (%)
0 1 0 1
10 373 340 33 9.8

Design Verification and Product Data for Alkaline Phosphatase opt. liquicolor 7/9
Rev. 007
 20 684 680 4 0.5
30 1034 1020 13 1.3
40 1361 1360 0 0.0
50 1684 1701 -16 -1.0
60 1992 2041 -49 -2.4
70 2312 2381 -69 -2.9
80 2618 2721 -103 -3.8
90 2935 3061 -126 -4.1
100 3272 3401 -129 -3.8

ALKALINE PHOSPHATASE opt. liquicolor Multipurpose reagent is specified to cover a linear range on an AU 480 as
indicated in section 3: up to 2000 U/l

Conclusion
All real time stability data for linearity confirm the shelf life
of 18 months for ALKALINE PHOSPHATASE opt. liquicolor Multipurpose reagent.

6 Interference
Interference by ascorbate, bilirubin, hemoglobin, triglycerides and lipemic sera 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.Cem. 1986, 32 470-5).

The results are summarised in the following tables.

Reagent start (2-shot)

Ascor. Result Dev. Bili. Result Dev. Hemo. Result Dev. Triglyc Result Dev. Lipem Result Dev.
mg/dl U/l % mg/dl U/l % mg/dl U/l % mg/dl U/l % mg/dl U/l %
0 209 100.0 0 199 100.0 0 196 100.0 0 158 100.0 0 194 100
2 197 94.1 4 198 99.3 50 193 98.8 250 164 104.0 100 194 99.7
4 199 95.6 8 197 99.0 100 194 99.3 500 164 104.1 200 195 100.4
6 199 95.2 12 202 101.5 150 192 98.2 750 161 101.9 300 195 100.7
8 204 97.4 16 198 99.4 200 185 94.8 1000 164 103.7 400 195 100.6
10 195 93.2 20 204 102.4 250 184 93.9 1250 163 103.5 500 197 101.6
12 195 93.3 24 194 97.5 300 182 93.3 1500 160 101.5 600 195 100.5
14 194 92.8 28 200 100.3 350 182 93.0 1750 162 102.9 700 194 99.9
16 194 92.6 32 194 97.3 400 182 93.0 2000 166 105.4 800 200 102.8
18 195 93.1 36 198 99.2 450 178 91.2 2250 170 107.5 900 199 102.5
20 192 91.9 40 195 98.0 500 179 91.7 2500 168 106.3 1000 197 101.3

Glick 2 1 2 2 1

Sample start (1-shot)

Ascor. Result Dev. Bili. Result Dev. Hemo. Result Dev. Triglyc Result Dev. Lipem Result Dev.
mg/dl U/l % mg/dl U/l % mg/dl U/l % mg/dl U/l % mg/dl U/l %
0 227 100 0 221 100 0 224 100 0 182 100 0 223 100
2 225 98.9 4 223 100.7 50 221 98.6 250 186 102.1 100 222 99.7
4 227 100.0 8 226 102.1 100 223 99.3 500 187 102.5 200 216 97.2
6 229 100.7 12 225 101.7 150 223 99.2 750 185 101.8 300 219 98.6
8 228 100.4 16 222 100.4 200 219 97.6 1000 187 102.8 400 216 97.0
10 223 98.1 20 224 101.2 250 215 95.9 1250 187 102.6 500 217 97.3
12 226 99.3 24 223 100.9 300 222 98.9 1500 189 103.9 600 219 98.2
14 229 100.6 28 228 103.2 350 222 98.7 1750 192 105.2 700 220 98.9
16 223 98.3 32 226 102.0 400 215 95.7 2000 188 103.1 800 221 99.2
18 227 99.9 36 229 103.5 450 218 97.2 2250 188 103.3 900 219 98.5
20 224 98.4 40 221 100.0 500 215 95.7 2500 192 105.5 1000 220 98.6

Glick 1 2 2 2 1

Design Verification and Product Data for Alkaline Phosphatase opt. liquicolor 8/9
Rev. 007
Conclusion: This test is not significantly influenced by ascorbate up to 20 mg/dl, bilirubin up to 40 mg/dl,
hemoglobin up to 500 mg/dl, triglycerides up to 2500 mg/dl and lipemic sera up to 1000 mg/dl.

7 Traceability
The ALKALINE PHOSPHATASE opt. liquicolor concentration has been verified with the reference material SRM 909 B.
The ALKALINE PHOSPHATASE opt. liquicolor revealed recovery well within the reproducibility limits of the method.
The method is traceable to SRM 909 B.

Design Verification and Product Data for Alkaline Phosphatase opt. liquicolor 9/9
Rev. 007