117
Clinica Chimica Acta, 60 (1975) 117-120 @ Elsevier Scientific Publishing Company,
Amsterdam
-
Printed
in The Netherlands
SHORT COMMUNICATION CCA 6921
HEPATITIS B ANTIGEN IN CLINICAL CHEMISTRY
H.H. KREUTZER
and J. RECHSTEINER
R. K. Ziekenverpleging, (Received
CONTROL SERA
October
Hiluersum
(The Netherlands)
21, 1974)
In recent studies [l--6] a substantial part of commercial control sera, for clinical chemistry as well as for serology and hematology, prepared from human blood was found positive for HB Ag (Table I). All authors used the same R.I.A. method, i.e. the Ausria 125 Test Kit (Abbott). Only a small number of these sera, however, gave positive reactions with other tests (counter electrophoresis, complement fixation and others) which are known to be less sensitive than the R.I.A. At about the same time the specificity of the Ausria 125 test has been questioned. Sgouris [7] incubated 20 positive sera with cavia serum and repeated the test. Only two of them were found to be positive again, so in the remaining sera antibodies must have been present which cross-reacted with antigens in the cavia antiserum to HB Ag. Prince et al. [8] reported that 48% of Ausria positive sera became negative after absorption with cavia serum. Moreover, after incubation with HB Ab only 21% of the Ausria positive sera were found to be negative in a second test. Meanwhile a new version of the Ausria kit (Ausria II) has become availaTABLE
I
FREQUENCY Only
Ref.
control
OF
HB
sera for
Ag-POSITIVE clinical
Authors
chemistry
CONTROL
SERA
FOUND
IN EARLIER
STUDIES
[l-61
are mentioned.
Year
Number
POS.
%
90
of sera tested
1
Ginsberg
2
Simon
et al.
3
Wetli
4
Watson
5
Vacca
6
Thamer
et al.
Present
study
et al. et al. et al. et al.
1972
10
9
1973
28
22
79
1973
46
28
61
1973
48
35
73
1974
20
11
55
1974
13
9
69
58
Mean:
70
26
45
118 TABLE HB Data HB
II
Ag
CONTENT
are sheep
names
OF
expressed
and
COMMERCIAL
as U/N
immunoglobulin the
batch
ratws and
numbers
Manufacturer
Trade
CONTROL (see
normal of
text).
The
sheep
commercial
AND sera
immunoglobulin. sera
U/N
SERA positive
ratio
are
available
in Ausria
PRIVATE
SERUM
were
also
tested
The
names
of
from
the
first
POOLS
after the
incubation
with
anti-
manufacturers.
the
trade
author.
Conclusiont
II test
HBAg
name Without
After
incubation
(1 + 1) with
present:
incubation
Commercial 1
2
control
Sheep
Normal
anti-HB
sheep
sera a
17.6
1.1
3.4
b
21.8
1.4
24.3
a
0.9
_
_
0.8
_
+
0.8 _
0.8
_._
0.9 3.2
1.3
1.1
_
_
1.0 0.9
3.5 _
_
2.0 1.7
_
0.8
_
_
_ _ + +
30.9
1.1
19.1
24.3
1.0
35.1
0.9
_
0.9 9.6
1.2
11.1
20.1
1.0
11.2
31.0
1.4
8.3
4.8
0.8
3.8 4.1
7.2
1.2
11.1
1.2
6.2
14.5
1.0
18.9
2.6
1.0
1.3 _
1.1 3.3
+ + + + +
1.4
*
1.8
0.8 0.9
_
2.5
1.2 __
2.5
1.0 9.4
1.1
8.2
8.5
6.9
1.2
1.4 _
24.0
1.4
23.6
20.1
1.2
10.8
16.2
1.1
8.7
+
13.1
+
+
1.2 _
1.1 20.5 0.6
1.5
*
119 TABLE
II
(rotltinucd)
Manufacturer
U/N
Trade
ratio
in AUsria
II test
Conclusiont
name
HBAg Without incubation
After Sheep
Private
serum
Laboratory
incubation
(1 + 1) with ~____~_
present:
Normal
anti-HB
sheep
IgG
I&
pools
1
2.7 30.6 5.1
1.5
2.2
2.3
11.6
+
0.9
5.2
+
-
1.5 1.1
+
_
_
Laboratory
2
1.2
Laboratory
3
25.7
1.6
7.7
Laboratory
4
40.9
1.2
19.6
+
6.4
1.2
2.1
+
+
0.9 1.4 _
Laboratory
5
0.9
Laboratory
6
0.8
-
Laboratory
7
1.0
-
Laboratory
8
Cutoff
_
1.3 15.1
f
_
value:
U/N
ratio
2
1.1
6.1
+
2.1.
ble. In this test the cavia antiserum to HB Ag has been replaced by a human one in order to exclude cross reactions with cavia antigens. We thought it appropriate to investigate a number of control sera by this improved test taking care to avoid false positive results. From the stocks of five different laboratories 42 commercial control sera were chosen at random and another 16 samples were taken from private serum pools in eight laboratories. On these 58 sera of human origin the Ausria test was carried out following the instructions of the manufacturer. In Table II the reactivity with anti-HB is expressed as the ratio of the net count per minute of the unknown sample and the mean net count of seven negative control sera (U/N ratio). The test is considered positive when the U/N ratio exceeds the value 2.1 (cutoff value). All sera in which results were obtained exceeding the cutoff value were retested after incubation with equal volumes of sheep anti-HB immunoglobulin as well as with normal sheep immunoglobulin. Incubation was carried out for one hour at 37°C followed by an overnight stay at 4°C. As can be seen from Table II, 28 sera were positive in the original test. When these sera were retested after incubation with anti-HB, in all cases the activity fell under the cutoff value, but not so after incubation with normal immunoglobulin, with the exception of two sera. These sera (* in Table II) exceeded the cutoff value in the original test only marginally. After correction for these possibly false-positive results the total number of HB Ag positive control sera is 26 (45%).
120
We found no difference between commercial control sera (45% positive) and private serum pools (44% positive). It seems likely that the batches of Ausria 125, used in the previous studies on control sera were more specific than those used by Sgouris and by Prince et al. The lower percentage of positive sera found in our investigation can be explained by the greater specificity of Ausria II compared with Ausria 125. HB Ag-positive specimens (blood, serum, urine, feces, semen) from patients form a real hazard for laboratory workers. Testing of such specimens cannot be avoided. Positive control sera are particularly dangerous because they will be handled daily by a great number of technicians. From this point of view, clinical chemists who are responsible for the safety in their laboratories, should preferably use control sera of animal origin or, if they consider using human sera, only those batches in which the absence of HB Ag is proven by the most sensitive and specific test available. In view of the worldwide use of commercial control sera international control seems also desirable. Private control sera, obtained by mixing the remnants of a great number of patient sera will often appear to be positive for HB Ag. They should be tested too, before use or prepared from the sera of a small number of people who are negative for this antigen. References 1
A.L.
Ginsberg
2
R.G.
Simon,
3
C.V.
Wetli,
4
D.
and L..4.
A.V.
Watson,
M.E.
Conrad,
Langhofer Heal
D.L.
and
Langbey,
New.
and J.B.
Miale.
I.L.
Engl.
E.J.
J. Med..
Hendricks, Am.
Christie,
J. Clin. M.N.
287
Clin.
(1972)
Chem..
Pathol.,
Islam.
59
1097 19
(1973)
(1973)
J. Bertrand
and
985 5
G. Vacca,
6
G. Thamer
C. Trovaty
7
G.T.
Sgouns.
8
A.M.
Prince.
and
and
U. Bartsch,
New
F. DiSiena. Artzl.
F:ngl. J. Med.,
B. Brotman,
Lab., 288
D. Jass and
Clin.
Chim.
Acta.
20
(1974)
29
(1973) H.I.
50 (1974)
289
i (1973)
1346
160 Kram,
Lancet.
221
684 J.E.
Banatvala.
Lancet.
i (1973)
Clinica Chimica Acta, 60 (1975) 117-120 @ Elsevier Scientific Publishing Company,
Amsterdam
-
Printed
in The Netherlands
SHORT COMMUNICATION CCA 6921
HEPATITIS B ANTIGEN IN CLINICAL CHEMISTRY
H.H. KREUTZER
and J. RECHSTEINER
R. K. Ziekenverpleging, (Received
CONTROL SERA
October
Hiluersum
(The Netherlands)
21, 1974)
In recent studies [l--6] a substantial part of commercial control sera, for clinical chemistry as well as for serology and hematology, prepared from human blood was found positive for HB Ag (Table I). All authors used the same R.I.A. method, i.e. the Ausria 125 Test Kit (Abbott). Only a small number of these sera, however, gave positive reactions with other tests (counter electrophoresis, complement fixation and others) which are known to be less sensitive than the R.I.A. At about the same time the specificity of the Ausria 125 test has been questioned. Sgouris [7] incubated 20 positive sera with cavia serum and repeated the test. Only two of them were found to be positive again, so in the remaining sera antibodies must have been present which cross-reacted with antigens in the cavia antiserum to HB Ag. Prince et al. [8] reported that 48% of Ausria positive sera became negative after absorption with cavia serum. Moreover, after incubation with HB Ab only 21% of the Ausria positive sera were found to be negative in a second test. Meanwhile a new version of the Ausria kit (Ausria II) has become availaTABLE
I
FREQUENCY Only
Ref.
control
OF
HB
sera for
Ag-POSITIVE clinical
Authors
chemistry
CONTROL
SERA
FOUND
IN EARLIER
STUDIES
[l-61
are mentioned.
Year
Number
POS.
%
90
of sera tested
1
Ginsberg
2
Simon
et al.
3
Wetli
4
Watson
5
Vacca
6
Thamer
et al.
Present
study
et al. et al. et al. et al.
1972
10
9
1973
28
22
79
1973
46
28
61
1973
48
35
73
1974
20
11
55
1974
13
9
69
58
Mean:
70
26
45
118 TABLE HB Data HB
II
Ag
CONTENT
are sheep
names
OF
expressed
and
COMMERCIAL
as U/N
immunoglobulin the
batch
ratws and
numbers
Manufacturer
Trade
CONTROL (see
normal of
text).
The
sheep
commercial
AND sera
immunoglobulin. sera
U/N
SERA positive
ratio
are
available
in Ausria
PRIVATE
SERUM
were
also
tested
The
names
of
from
the
first
POOLS
after the
incubation
with
anti-
manufacturers.
the
trade
author.
Conclusiont
II test
HBAg
name Without
After
incubation
(1 + 1) with
present:
incubation
Commercial 1
2
control
Sheep
Normal
anti-HB
sheep
sera a
17.6
1.1
3.4
b
21.8
1.4
24.3
a
0.9
_
_
0.8
_
+
0.8 _
0.8
_._
0.9 3.2
1.3
1.1
_
_
1.0 0.9
3.5 _
_
2.0 1.7
_
0.8
_
_
_ _ + +
30.9
1.1
19.1
24.3
1.0
35.1
0.9
_
0.9 9.6
1.2
11.1
20.1
1.0
11.2
31.0
1.4
8.3
4.8
0.8
3.8 4.1
7.2
1.2
11.1
1.2
6.2
14.5
1.0
18.9
2.6
1.0
1.3 _
1.1 3.3
+ + + + +
1.4
*
1.8
0.8 0.9
_
2.5
1.2 __
2.5
1.0 9.4
1.1
8.2
8.5
6.9
1.2
1.4 _
24.0
1.4
23.6
20.1
1.2
10.8
16.2
1.1
8.7
+
13.1
+
+
1.2 _
1.1 20.5 0.6
1.5
*
119 TABLE
II
(rotltinucd)
Manufacturer
U/N
Trade
ratio
in AUsria
II test
Conclusiont
name
HBAg Without incubation
After Sheep
Private
serum
Laboratory
incubation
(1 + 1) with ~____~_
present:
Normal
anti-HB
sheep
IgG
I&
pools
1
2.7 30.6 5.1
1.5
2.2
2.3
11.6
+
0.9
5.2
+
-
1.5 1.1
+
_
_
Laboratory
2
1.2
Laboratory
3
25.7
1.6
7.7
Laboratory
4
40.9
1.2
19.6
+
6.4
1.2
2.1
+
+
0.9 1.4 _
Laboratory
5
0.9
Laboratory
6
0.8
-
Laboratory
7
1.0
-
Laboratory
8
Cutoff
_
1.3 15.1
f
_
value:
U/N
ratio
2
1.1
6.1
+
2.1.
ble. In this test the cavia antiserum to HB Ag has been replaced by a human one in order to exclude cross reactions with cavia antigens. We thought it appropriate to investigate a number of control sera by this improved test taking care to avoid false positive results. From the stocks of five different laboratories 42 commercial control sera were chosen at random and another 16 samples were taken from private serum pools in eight laboratories. On these 58 sera of human origin the Ausria test was carried out following the instructions of the manufacturer. In Table II the reactivity with anti-HB is expressed as the ratio of the net count per minute of the unknown sample and the mean net count of seven negative control sera (U/N ratio). The test is considered positive when the U/N ratio exceeds the value 2.1 (cutoff value). All sera in which results were obtained exceeding the cutoff value were retested after incubation with equal volumes of sheep anti-HB immunoglobulin as well as with normal sheep immunoglobulin. Incubation was carried out for one hour at 37°C followed by an overnight stay at 4°C. As can be seen from Table II, 28 sera were positive in the original test. When these sera were retested after incubation with anti-HB, in all cases the activity fell under the cutoff value, but not so after incubation with normal immunoglobulin, with the exception of two sera. These sera (* in Table II) exceeded the cutoff value in the original test only marginally. After correction for these possibly false-positive results the total number of HB Ag positive control sera is 26 (45%).
120
We found no difference between commercial control sera (45% positive) and private serum pools (44% positive). It seems likely that the batches of Ausria 125, used in the previous studies on control sera were more specific than those used by Sgouris and by Prince et al. The lower percentage of positive sera found in our investigation can be explained by the greater specificity of Ausria II compared with Ausria 125. HB Ag-positive specimens (blood, serum, urine, feces, semen) from patients form a real hazard for laboratory workers. Testing of such specimens cannot be avoided. Positive control sera are particularly dangerous because they will be handled daily by a great number of technicians. From this point of view, clinical chemists who are responsible for the safety in their laboratories, should preferably use control sera of animal origin or, if they consider using human sera, only those batches in which the absence of HB Ag is proven by the most sensitive and specific test available. In view of the worldwide use of commercial control sera international control seems also desirable. Private control sera, obtained by mixing the remnants of a great number of patient sera will often appear to be positive for HB Ag. They should be tested too, before use or prepared from the sera of a small number of people who are negative for this antigen. References 1
A.L.
Ginsberg
2
R.G.
Simon,
3
C.V.
Wetli,
4
D.
and L..4.
A.V.
Watson,
M.E.
Conrad,
Langhofer Heal
D.L.
and
Langbey,
New.
and J.B.
Miale.
I.L.
Engl.
E.J.
J. Med..
Hendricks, Am.
Christie,
J. Clin. M.N.
287
Clin.
(1972)
Chem..
Pathol.,
Islam.
59
1097 19
(1973)
(1973)
J. Bertrand
and
985 5
G. Vacca,
6
G. Thamer
C. Trovaty
7
G.T.
Sgouns.
8
A.M.
Prince.
and
and
U. Bartsch,
New
F. DiSiena. Artzl.
F:ngl. J. Med.,
B. Brotman,
Lab., 288
D. Jass and
Clin.
Chim.
Acta.
20
(1974)
29
(1973) H.I.
50 (1974)
289
i (1973)
1346
160 Kram,
Lancet.
221
684 J.E.
Banatvala.
Lancet.
i (1973)
![[Serological hepatitis B characteristics in clinical laboratory control sera].](/assets/img/hold.png)
