Design Verification

auto-B-D
auto liquicolor
System Reagent for HumaStar 600 (HS600SR)

C

1 Introduction .................................................................................................................................................................. 2
2 Imprecision .................................................................................................................................................................... 2
3 Linearity and Detection Limit ...................................................................................................................................... 2
3.1 Linearity ....................................................................................................................................................................................... 2
3.2 Detection Limit ......................................................................................................................................................................... 3
4 Comparison of methods ............................................................................................................................................... 3
5 Stability, Accelerated Stress ........................................................................................................................................ 5
5.1 Linearity ....................................................................................................................................................................................... 5
5.2 Recovery of control sera ........................................................................................................................................................ 6
6 Real Time Stability ........................................................................................................................................................ 6
7 On Board stability / Calibration stability .................................................................................................................... 6
7.1 Recovery of control sera ........................................................................................................................................................ 6
7.2 Calibration stability ................................................................................................................................................................. 7
8 Interferences ................................................................................................................................................................. 7
9 Traceability .................................................................................................................................................................... 7

Form: 4.2-01.06-04
Rev. 007 | valid of 26.11.2013 1/7
1 Introduction
The performance characteristics of the auto-BILIRUBIN-D liquicolor HS600SR 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 the auto-BILIRUBIN-D liquicolor HS600SR was calculated from six
determinations on five consecutive days using reagent lot # H022 (expiry 2006-12). Low, medium and high level
control sera (lot # CE 127 A, CE 127 B and CE 127 C) were employed as sample material. Acceptance criteria were a
max. CV of 4% (intra-assay) and 5% (inter-assay).

Mean analyte concentration Inter-assay Intra-assay

(mg/dl) SD (mg/dl) %CV SD (mg/dl) %CV
0.59 0.019 3.13 0.009 1.48
0.92 0.022 2.38 0.015 1.65
5.24 0.071 1.35 0.066 1.25

Conclusion: The auto-BILIRUBIN-D liquicolor HS600SR assay shows acceptable precision.

3 Linearity and Detection Limit

3.1 Linearity
The linearity of the auto-BILIRUBIN-D liquicolor HS600SR, lot 0042/0001A was controlled by employing a high
concentrated pool serum successively diluted with phys. saline. The analysed concentrations were compared with
the theoretical concentrations obtained from a linear regression. Deviations of measured against theoretical
concentrations are expressed in absolute and relative values. Linearity was assumed within an accepted deviation
of ± 10% starting at high pool content of 10%.

High Pool Analytical Data Regressed Data Deviation from Regression Line
Content (%) mg/dl mg/dl mg/dl (%)
0 0.1 0.5 -0.40
10 1.3 1.5 -0.14 -9.6
20 2.5 2.4 0.07 2.9
30 3.5 3.4 0.14 4.1
40 4.5 4.3 0.15 3.5
50 5.6 5.3 0.29 5.5
60 6.4 6.3 0.09 1.4
70 7.3 7.2 0.12 1.7
80 8.2 8.2 -0.02 -0.2
90 9.0 9.1 -0.10 -1.1
100 9.8 10.1 -0.29 -2.9

Design Verification and Product Data for auto-Bilirubin-D HS600SR

Rev. 007 2/7
Conclusion: The auto-BILIRUBIN-D liquicolor HS600SR is linear up to 10 mg/dl.

3.2 Detection Limit

The detection limit has been evaluated from a 20-fold repeat of a zero sample (saline at 0.9% NaCl) according to
IUPAC recommendations, using kit lot # H032 on HumaStar 600. It was calculated on base of absolute mean + 3 SD
(mean of 0.01 mg/dl and SD of 0.01 mg/dl). From these results the detection limit has been set to 0.1 mg/dl.

3.3 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. The determinations of each control serum
have been performed with the auto-BILIRUBIN-D liquicolor HS600SR, lot 0042/001. The mean values obtained with
fresh reagent have been calculated and compared with the target values of the respective control sera (accepted
mean deviation of ± 10%). The recovery has to be in the target range.

CONTROL SERUM RECOVERY auto-BILIRUBIN-D reagent

8 d 37 °C
Control LOT Target Range Result Deviation
serum mg/dl mg/dl mg/dl (%)
HUMATROL N 0001 0.3 0.22 - 0.38 0.33 10.0
HUMATROL P 0002 3.63 2.69 - 4.58 3.47 -4.4
SERODOS 0002 1.31 0.97 - 1.65 1.25 -4.6
SERO.Plus 0001 2.22 1.64 - 2.80 2.23 0.5
Precinorm 164231 0.79 0.601 - 0.979 0.76 -3.8
Precipath 163957 2.39 1.82 - 2.96 2.37 -0.8
Olympus 1 031 1.11 0.82 - 1.40 1.27 14.4
Olympus 2 030 4.73 3.50 - 5.96 4.91 3.8
Autocal H014 3.49 3.55 1.7
Mean 2.22 2.24 0.9

4 Comparison of methods
The auto-BILIRUBIN-D liquicolor HS600SR lot 0013 run on HumaStar 600 has been compared against the BILIRUBIN-
D reagent (Beckman) lot 9967 method measured on Olympus AU 400. Control sera have been employed in the
comparison (N=74). The results have been evaluated by a non-parametric regression model according to Bablok &
Passing, Acceptance criteria were r > 0.95 and a slope between 0.95 and 1.05 and could be described as follows:

Design Verification and Product Data for auto-Bilirubin-D HS600SR

Rev. 007 3/7
 r = 0.9917
Y= 0.9880 X 0.0493
Xmean = 1.48 mg/dl
Ymean = 1.48 mg/dl

Sample-
Sample- Mean Reference Mean Test
no. Beckman Olympus AU 400 HumaStar 600 SR
1 0.59 0.64
2 0.66 0.61
3 0.39 0.30
4 0.08 0.19
5 0.72 0.71
6 0.13 0.18
7 0.94 0.82
8 0.23 0.30
9 0.10 0.16
10 0.67 0.88
11 1.16 1.19
12 0.06 0.11
13 0.14 0.16
14 0.60 0.50
15 0.08 0.15
16 0.63 0.73
17 0.08 0.11
18 0.11 0.16
19 0.92 0.84
20 0.15 0.18
21 1.02 1.07
22 0.09 0.12
23 0.72 0.79
24 0.20 0.25
25 2.23 2.08
26 0.45 0.51
27 0.06 0.26
28 0.07 0.22
29 0.46 0.43
30 0.10 0.14
31 0.17 0.22
32 1.06 1.14
33 0.34 0.30
34 1.89 2.00
35 1.79 1.82
36 0.55 0.67
37 0.60 0.71
38 0.67 0.70
39 1.08 1.21
40 0.52 0.50
41 0.55 0.51
42 1.11 1.18
43 0.51 0.56
44 0.66 0.70
45 0.52 0.54
46 3.53 3.46
47 3.76 3.89
48 3.60 3.55
49 4.85 4.79
50 3.16 2.22
51 3.50 3.66
52 2.05 2.06
53 3.58 3.76
54 3.51 3.53
55 3.61 3.43
56 5.64 5.57
57 5.17 4.65
58 3.08 3.15
59 1.39 1.43
60 1.65 1.76
61 1.59 1.47
62 3.56 3.47

Design Verification and Product Data for auto-Bilirubin-D HS600SR

Rev. 007 4/7
 63 3.61 3.77
64 0.09 0.14
65 2.81 3.18
66 1.57 1.67
67 3.99 4.29
68 2.43 2.34
69 1.68 1.87
70 2.23 2.35
71 2.32 1.87
72 1.21 0.92
73 2.84 2.51
74 2.02 1.59

Results from a non-parametric regression analysis acc. to Passing & Bablok are shown in the graph below.

Conclusion: Both methods showed a good agreement and no significant deviation could be observed with
any specific sample.

5 Stability, Accelerated Stress

5.1 Linearity
The linearity of the auto-BILIRUBIN-D liquicolor HS600SR was controlled according to the procedure already
described in 3.1. The reagents were stressed in original HumaStar 600 plastic bottles 14 days at 37°C, lot
0042/0001, expiry date 2013-05.

Linearity stress 14 days 37°C

High Pool Analytical Data Regressed Data Deviation from Regression Line
Content (%) mg/dl mg/dl mg/dl %
0 0.1 0.5 -0.40
10 1.3 1.5 -0.13 -8.9
20 2.5 2.4 0.09 3.7
30 3.5 3.4 0.16 4.7
40 4.5 4.3 0.20 4.6
50 5.5 5.3 0.24 4.5
60 6.4 6.3 0.14 2.2
70 7.4 7.2 0.17 2.4

Design Verification and Product Data for auto-Bilirubin-D HS600SR

Rev. 007 5/7
 80 8.2 8.2 -0.02 -0.2
90 9.0 9.1 -0.19 -2.1
100 9.4 10.1 -0.68 -6.7

Conclusion: The test is specified to cover a linear range up to 10 mg/dl as indicated in section 3.1. The above
results clearly confirm that the test achieves its specification after accelerated stress.

5.2 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. The determinations of each control serum
have been performed with the auto-BILIRUBIN-D liquicolor HS600SR, lot 0042/001. The mean values obtained with
fresh and stressed reagent have been calculated and compared with the fresh values of the respective control sera
(accepted deviation of ± 10%).

CONTROL SERUM RECOVERY auto-BILIRUBIN-D auto-BILIRUBIN-D reagent

reagent fresh 14 d 37 °C
Control LOT Target Range Result Result Deviation
serum mg/dl mg/dl mg/dl mg/dl (%)
HUMATROL N 0001 0.3 0.22 - 0.38 0.33 0.32 -3.0
HUMATROL P 0002 3.63 2.69 - 4.58 3.47 3.44 -0.9
SERODOS 0002 1.31 0.97 - 1.65 1.25 1.22 -2.4
SERO.Plus 0001 2.22 1.64 - 2.80 2.23 2.13 -4.5
Precinorm 164231 0.79 0.601 - 0.979 0.76 0.76 0.0
Precipath 163957 2.39 1.82 - 2.96 2.37 2.28 -3.8
Olympus 1 031 1.11 0.82 - 1.40 1.27 1.28 0.8
Olympus 2 030 4.73 3.50 - 5.96 4.91 4.84 -1.4
Autocal H014 3.49 3.55 3.4 -4.2
Mean 2.22 2.24 2.19 -2.2

Conclusion: Accelerated stress data are indicative for a real-time stability of 24 months as determined for the
corresponding multipurpose reagent, supporting the claimed shelf life of 18 months.

6 Real Time Stability

Real-time studies for auto-BILIRUBIN-D liquicolor HS600 are ongoing.

7 On Board stability / Calibration stability

The auto-BILIRUBIN-D liquicolor HS600SR have been stored unscrewed on the HumaStar 600 analyzer for more
than 60 days. The calibration have been performed with auto-BILIRUBIN-D liquicolor HS600SR on the first day.

7.1 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. The calibration has been done on the first
day. The determinations of each control serum have been performed with the auto-BILIRUBIN-D liquicolor HS600SR
fresh and after 61 days on board. The mean value has been calculated and compared with the fresh mean of the
respective control sera (accepted deviation ± 10%).

CONTROL SERUM RECOVERY auto-BILIRUBIN-D auto-BILIRUBIN-D reagent

reagent fresh 61 d on board
Control LOT Target Range Result Result Deviation
serum mg/dl mg/dl mg/dl mg/dl (%)
HUMATROL N # 021 0.41 0.30 – 0.52 0.45 0.47 3.98
HUMATROL P # 019 0.87 0.64 – 1.10 0.82 0.96 16.2
SERODOS # 6870 0.59 0.44 – 0.74 0.66 0.52 -21.8
SERO.Plus # 6798 1.30 0.96 – 1.64 1.39 1.37 -1.87
AUTOCAL # H012 1.37 1.40 1.36 -2.43
Mean 0.91 0.95 0.93

Design Verification and Product Data for auto-Bilirubin-D HS600SR

Rev. 007 6/7
Conclusion: The on board and calibration stability of the auto-BILIRUBIN-D liquicolor HS600SR is confirmed
for a minimum of 60 days.

7.2 Calibration stability

For the determination of the calibration stability the same procedure and reagents as in 7.1 were used. After
calibration on day 14 the concentration of control sera were determined. Seven days later the measurement were
repeated with the existing calibration. A deviation of ± 5% for every control serum is accepted as a stable
calibration .

CONTROL SERUM RECOVERY Day 14 Day 21

Control LOT Target Range Result Deviation
serum mg/dl mg/dl mg/dl (%)
HUMATROL N # 021 0.41 0.30 – 0.52 0.48 0.46 -3.8
HUMATROL P # 019 0.87 0.64 – 1.10 0.90 0.89 -1.6
SERODOS # 6870 0.59 0.44 – 0.74 0.63 0.61 -2.6
SERO.Plus # 6798 1.30 0.96 – 1.64 1.44 1.40 -3.1
AUTOCAL # H012 1.37 1.39 1.38 -0.6

Mean 0.91 0.97 0.95 -2.3

Conclusion: The calibration stability of the auto-BILIRUBIN-D liquicolor HS600SR is confirmed for a minimum
of 7 days.

8 Interferences
Interference by hemoglobin, lipemia (intralipid) and ascorbic acid have been studied by adding known amounts of
the potentially interfering substance to a known sample.
Recoveries have been analysed according to the method of Glick et al. (Clin.Chem. 1986, 32 470-5).
Criterion Recovery within ± 10 % of initial value.
The results are summarised in the following table.

Hemoglobin Analyt. Result Result Intralipid Analyt. Result Result % Ascorbate Analyt. Result Result %
mg/dl mg/dl % mg/dl mg/dl mg/dl mg/dl
0 0.81 100 0 0.94 100 0 0.91 100.0
50 0.24 29.0 100 1.04 110.6 2 0.91 99.5
100 0.09 11.1 200 0.61 64.4 4 0.92 100.5
150 0.07 8.6 300 0.07 7.4 6 0.89 97.3
200 0.08 9.3 400 8 0.91 100.0
250 0.08 9.9 500 10 0.90 98.4
300 0.09 11.1 600 12 0.90 98.9
350 0.11 13.0 700 14 0.90 98.9
400 0.12 14.8 800 16 0.89 97.8
450 0.14 17.3 900 18 0.90 98.4
500 0.15 18.5 1000 20 0.91 99.5

Glick 4 n.a 1

Conclusion: This test is not significantly influenced by ascorbate < 20 mg/dl. Lipemic samples strongly
interfere and should not be used, there is poor correlation between intralipid and triglycerides. A
Glick value of 4 for hemoglobin indicates a strong interfering effect. Hemolytic samples should
be avoided.

9 Traceability
The auto-BILIRUBIN-D liquicolor HS600SR calibrated with AUTOCAL revealed recovery well to standardised
competitor methods within the reproducibility limits of the method. Reference material for direct bilirubin is not
available.

Design Verification and Product Data for auto-Bilirubin-D HS600SR

Rev. 007 7/7