INVESTIGATION OF LOW-LEVEL RUBELLA IgG RESULTS

Rubella Reporting –
Where did we go wrong and what are
we doing about it?
Dimech W
NRL, Melbourne, Australia
31st Annual NRL Workshop on Infectious Disease
Melbourne, 30 July 2014
Outline of presentation
Rubella virus
Vaccination
WHO International Standard
History of testing
Determination of immune/non-immune cut-off
Lack of standardisation
Principles of standardisation
Summary of the problem
What’s next
Rubella virus
Single stranded RNA;
Genus: rubivirus;
Family: Togaviridae
Three structural polypeptides
Nucleocapsid, (C polypeptide chain)
El glycopolypeptide (predominant reactivity)
E2a glycopolypeptide
E2b glycopolypeptide
Diagnostic Virology Protocols
Methods in Molecular Medicine™ Volume 12, 1998, pp 143-157
Rubella virus
Virus in nasopharynx
HAI
Viraemia
R
IgG
A
S
H
IgM
Infection
1
2
3
Weeks
4
5
6
Vaccination
1971- Vaccination of 10-14 yearold girls started
1989- MMR vaccination of all
infants (12-15 Mo)
1994- Vaccination of both boys
and girls (10 –16 years)
Immune response to vaccination
is often weaker than that found in
wild type infection
Vaccination
Francis, B. Am. J. Pub. Health. 2003: 93. No 8. 1274-6.
Vaccination
Courtesy of Hans Zaaijer, Sanquin, The Netherlands
WHO International Standard
1966- First International Reference Preparation of
Anti-Rubella Serum was established from a pool of
convalescent human sera
1968- The preparation had lost potency and was
discontinued
1970- Second International Reference Preparation
of Anti-Rubella Serum (RUBS) was established
RUBS prepared from normal human
immunoglobulin
1988- Name changed to second International
Standard for Anti-Rubella Serum, Human
WHO International Standard
1993- A replacement for RUBS was required
1995- A third International Standard (proposed)
(RUBI-1-94) prepared by Statens Serum Institute
Prepared from human immunoglobulin,
(BS/94.1762) with equal volume of saline
(lyophilised) – polyclonal antibodies
1995- Calibration of RUBI-1-94 performed by
testing in parallel with RUBS
1996- Renamed the First International Standard for
Anti-Rubella
WHO International Standard
Tested by 11 labs, 7 countries using EIA and HAI
Potency estimated as 1600 IU/mL using parallel
line statistics
IFU states
“Use of immunoglobulin preparations as a reference
material for immunoassays is not an ideal solution”
“Study has almost been completed”
Study results never reported but referenced in an
“unpublished 47th meeting of the WHO Expert
Committee on Biological Standardisation” (1996)
Used to calibrate almost all commercial assays
History of testing
Assay
Viral neutralisation
Units
Target
titre
Total Ig
Haemagglutination inhibition titre
Total Ig
Latex agglutination
titre
Total Ig
Immunofluorescence
titre
IgG
Single radial diffusion
IU/mL
Total Ig
Microtire plate EIA
IU/mL
IgG
Automated EIA (viral lysate) IU/mL
IgG
Automated EIA
(recombinant)
Specific
epitopes
IU/mL
Determination of immune / nonimmune cut-off
Initial studies on HAI and neutralisation assays
1978- Bradstreet suggested minimum titre be 2448 IU (HAI -1:16-1:20)
Cut-off of 15 IU/mL recommended by
(NCCSL/CSLI; 1985) and UK PHLS working
party (1988)
1987- IMx cut-off 10 IU/mL (Abbott)
1988- Reviewed cut-off was 10 IU/mL (CDC)
All reports acknowledge false positive and
negative results associated with cut-off
ARCHITECT
AxSYM
Elecsys
VIDAS
Vitros
Microparticles
Microparticles
Magnetic beads
Solid Phase
Receptacles (SPR)
Wells
Antigen
Partially purified
rubella virus
Partially purified
rubella virus
(strain HPV77)
Rubella-like
particles and
recombinant E1
antigen
Rubella antigen
(strain MR 383)
UV-treated rubella
antigen from cell
culture
Detection system
Chemiluminescence
Methylumbelliferyl
immunofluorescence
Chemiluminescence
Methylumbelliferyl
immunofluorescence
Luminescence
Number of
calibrators
6
6
2
1*
Four parameter
logistic curve
Calibration range
(IU/mL)
0 - 500
0 - 500
0.17 - 500
0 - 250
0 - 350
Standard
WHO standard 1st
International Standard
(RUB-1-94)
WHO standard
(not specified)
WHO standard 1st
International Standard
(RUB-1-94)
WHO standard 1st
International Standard
(RUB-1-94)
WHO standard 1st
International Standard
(RUB-1-94)
Negative range
(IU/mL)
<4.9
<5.0
<10
<5.0
<9.99
Equivocal range
(IU/mL)
(grey zone)
5.0-9.9
5.0-9.9
NA
5.0-10.0
10.0 - 14.9 **
Positive range
(IU/mL)
>10.0
>10.0
>10
>10.0
>15.0
Method
Solid Phase
* In addition to Master calibration;
** Low positive
Lack of standardisation
95% CI Value (IU/mL)
Comparison of results reported by
eight rubella IgG assays
180
160
140
120
100
80
60
40
AxSYM
A
Beckman
B
Centaur
C
Enzygnost
D
Immulite
AssayE
Liason
F
Sorin
G
Vidas
H
Assays
Dimech, W. et. al. 2008. Evaluation of eight anti-rubella virus immunoglobulin G immunoassays that report
results in international units per milliliter. J. Clin. Micro. 46: 1955-1960.
Investigation into low-level
rubella IgG results
Investigation into the level
of correlation between lowlevel test results reported
by five rubella IgG
immunoassays by
comparing these results
with a status determined
by HAI and western blot
testing
Dimech et. al. Clin. Vaccine Immunol. 2013, 20(2):255.
Investigation into low-level
rubella IgG results
One hundred routine clinical samples having
low-level rubella IgG test results (<40 IU/mL) on
each of five different anti-rubella IgG
immunoassays were collected
The immunosassays were:
Abbott ARCHITECT
Abbott AxSYM
bioMerieux VIDAS*
Ortho Vitros
Roche Elecsys
* FDA approved product (30 226)
Investigation into low-level
rubella IgG results
Rubella IgG status
Assay
Negative
Positive
Assay test result
Assay test result
Negative
Equivocal
Positive
Negative Equivocal Positive
Total
Number
Samples
Abbott ARCHITECT
13
5
5
1
7
69
100
Abbott AxSYM
6
2
1
2
5
84
100
bioMerieux VIDAS
6
6
18
70
100
Ortho Vitros
29
2
6
7
55
100
Roche Elecsys
29
15
5
51
100
1
RV-IgG evaluation 2013
∗ 325 pretested-negative RV-IgG samples
(from France, Italy and Germany) were tested with 9 assays:
∗
∗
∗
∗
∗
∗
∗
∗
∗
Immuno-blot Mikrogen
DxI Beckmann-Coulter
Architect Abbott
VIDAS bioMérieux
Enzygnost Siemens
LXL Diasorin
Cobas 6000 Roche
Centaur Siemens
Serion
Christelle VAULOUP-FELLOUS
National Reference Laboratory for Rubella
Virology department, Groupe Hospitalier
Paris-Sud
Medecine Faculty Paris-Sud 11 University,
France
Results (1)
Immunoblot
Mikrogen
DxI
Architect
VIDAS
Enzygnost
LXL
Cobas 6000
Centaur
BeckmannCoulter
Abbott
bioMérieux
Siemens
Diasorin
Roche
Siemens
Serion
Negative
134/325
41%
-
207/325
64%
202/325
62%
152/325
47%
209/325
64%
135/325
158/325
48%
215/325
66%
Equivocal
-
-
107/325
33%
58/325
18%
49/325
15%
84/325
26%
-
51/325
16%
88/325
27%
Positive
191/325
59%
-
11/325
3%
65/325
20%
124/325
38%
32/325
10%
190/325
58%
116/325
36%
22/325
7%
Results (2)
IBlot
DxI
Architect
VIDAS
Enzygnost
LXL
Cobas 6000
Centaur
BeckmannCoulter
Abbott
bioMérieux
Siemens
Diasorin
Roche
Siemens
E: 10-14
E: 5-9
E: 10-15
E: 5-6
E: 5-9
N<10
E: 5-10
Serion
E: 10-20
P
11,1
E
1,8
N
13
E
16
P
21,9
P
4,3
N
42,1
P
28,4
P
P
12,8
E
4,3
N
13
E
6
E
5,4
E
11,6
P
11,1
P
7,36
N
P
12,2
E
4,1
N
11
E
5
E
8,8
E
10,5
P
25,1
P
14,5
E
P
9,4
N
5
E
10
E
6
E
3,5
N
60,4
P
10,7
P
8,11
N
P
9,8
N
7,6
E
13
E
8
P
5,5
E
5
N
11,7
P
10,8
E
P
7,7
N
4,8
N
9
N
5
E
6,3
E
61,1
P
13,3
P
9,35
N
P
6,8
N
4,2
N
7
N
5
E
<3
N
11,8
P
9,3
E
6,1
N
P
8,9
N
5
E
14
E
8
P
5,7
E
41,2
P
17,1
P
10,6
E
P
8,3
N
4,8
N
11
E
8
P
8,8
E
11,4
P
13,6
P
12,1
E
P
12
E
4,1
N
12
E
7
P
8,6
E
7,7
N
23,5
P
12,5
E
P
12,2
E
7
E
10
E
13
P
4,9
N
>500
P
14,1
P
10,8
E
P
9,5
N
6,1
E
12
E
8
P
4,4
N
19,2
P
7,4
E
11,4
E
Summary of results of a QC
sample calibrated at 10 IU/mL
Assay
n
Mean
SD
CV (%)
Abbott ARCHITECT Rubella IgG CMIA
2386
12.64
1.20
9.49
Abbott AxSYM Rubella IgG MEIA
2728
12.11
2.54
20.99
bioMerieux VIDAS RUB IgG II ELFA
47
22.51
2.04
9.00
Ortho Vitros Rubella IgG Assay
419
27.25
2.88
10.57
Roche Elecsys Rubella IgG ECLIA
300
28.17
1.29
4.58
Siemens IMMULITE 2000 Rubella Quantitative IgG CLEIA
492
14.66
2.06
14.08
Siemens ADVIA Centaur Rubella G Assay
1881
21.62
3.43
15.85
Siemens Enzygnost Anti-Rubella IgG EIA
46
58.18
4.25
7.30
Principles of standardisation
In laboratory medicine, the exact definition of the
analyte, its biological and clinical function, and the
influence of the matrix are crucial elements when
establishing a Reference Method
The Reference Method (the highest possible level
in the metrologic hierarchy) needs to be specific for
the defined analyte
The starting points in the preparation of a Certified
Reference Material (CRM) are the pure or purified
analytes from human origin and the matrix
Muller, Clinical Chemistry 46, No. 12, 2000
Principles of standardisation
When such approaches are used for
analytes measured by immunoassays,
they are not accurate, not traceable, and not
in agreement with the principles of metrology.
When an immunoassay is used to certify the
value of a CRM, exact definition and
characterization of the analyte and the
reagent (including the antibody) are essential
for international acceptability of the values
assigned.
Muller, Clinical Chemistry 46, No. 12, 2000
Comparison of primary and
secondary standards
The problem
The problem
Information regarding the preparation of the
standard is lacking
Preparation of the standard does not comply
with metrologic principles
Initial testing of the standard was with HAI and
first generation EIAs, not reference methods
Immune cut-off of 10 IU/mL is debatable
Vaccination programs have resulted in more
low-level rubella IgG results
The low level results (<40 IU/mL) reported by
immunoassays are difficult to interpret
The problem
New generation assays have a range of
performance characteristics and are more
analytically sensitive compared with HAI
There is variation in the quantitative results
reported by immunoassays even though they use
the same standard
Incorrect results can lead to CRS or unnecessary
termination of pregnancy
What is next?
There is a move to abandon the reporting of
rubella IgG in IU/mL
Collaboration to develop a panel of anti-rubella
IgG negative samples
Samples, derived from multiple countries, will be
negative in a range of immunoassays and an
immunoblot
Available for purchase by manufacturers and used
to determine assay cut-off
Support from industry, WHO and European
virologists

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