Clinica Chimica Acta, 67 (1976) l-5 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7100
AN IMPROVED MANIFOLD OESTROGEN ASSAY
R.A. MOORE, Nuffield
(Received
DESIGN FOR RAPID AUTOMATED
M. RYAN and W.A.F. PENFOLD
Department
September
of Clinical
Biochemistry,
Radcliffe
Infirmary,
Oxford
OX2 6HE
(U.K.)
24, 1975)
Summary
A modified “debubbler-cactus” provides for a change from air to chloroform segmentation in an automated fluorimetric oestrogen assay. The reduction in dead space allows the rate of sampling to be increased to 60 per hour with good precision and acceptable carry-over.
Introduction
In response to the increased demand for urinary oestrogen analysis as a monitor of foetal-placental well-being, a number of automated methods became available to cope with the load in the late 1960s. The fastest rates of analysis were of the order of 20 per hour (Fournier et al., 1966 [2] ; Strickler et al., 1967 [9] ; Van Kessel et al., 1969 [4] ; Muir et al., 1969 [6] and 1971 [7] ; Lee and Htihnel, 1971 [ 51, and Hainsworth and Hall, 1971 [3]), although Campbell and Gardner (1971) [l] proposed a method which yielded a rate of 30 per hour. This paper presents a modification in the manifold design of Ryan and Gray (1975) [8], which allows analysis rate to be increased to 60 per hour without loss of precision or increase in carry-over. Materials and methods
Reagents All reagents were A.R. grade and were purchased from BDH Chemicals, Poole, Dorset. Quinol-sulphuric acid: 15 g of quinol are shaken with 350 ml of distilled water, and 650 ml of cone HzS04 are added rapidly enough to cause boiling. When cooled the solution is made up to 1 litre with distilled water. Chloroform/nitrophenol: 20 g/l of 4-nitrophenol in chloroform.
2
Diluent: 3.75 g/l Brij 35 in distilled water for the conventional manifold, and 1.25 g/l Brij 35 in the improved system. These concentrations gave the best phase separation. Twenty-four hour urine samples were collected into bottles containing a small amount of thiomersal as preservative. The volurnc~ was recorded and where necessary the samples were diluted to 2 I with water. Ten ml aliquots were taken for pretreatment with sodium borohydride to eradicate glucose interference (Ryan and Gray, 1975 [ Sj ). Standard Autoanalyzer equipment (Technicon Instruments Corp., Tarrytown, N.Y. 10591) was used, with the exception of a Newton Sampler with variable sample and wash times (Burkard Scientific Sales Ltd., Woodcock Hill, Rickmansworth, Her&. WD3 1PJ) and a Fluorimeter Mk IV (Locarte Co., 8 Wendell Road, London, W12) equipped with a thallium light source. At various rates of sampling carry-over was determined by sampling a 10 mg/Z 1 standard immediately after a 40 mg/2 1 standard. The average increase in the low standard over three such cycles was expressed as a percentage of top standard. Precision was determined by repeated sampling of t,wo urines at levels (average) of 6.5 mg/2 1 and 32 mg/2 1. The precision of the method is expressed as coefficient of variation (C.V.).
The essential difference between the conventional manifold (Fig. 1) and the improved design (Fig. 2) is the direct injection of the reacted sample (which has passed through a heating bath and cooling coil) into the nitrophenol/chloroform
CONVENTIONAL
MANIFOLD HEATING
BATH
PUMP ml/min
To.
SAMPLE
! 1.601
QUINOL-H2S04
10.42: .
.
I
I
I Il.201
I
I
I t
Fig.
1. Conventional
manifold.
I I I
AIR
DILUENT
1
IMPROVED
MANIFOLD PUMP HEATING
ml/min
BATH
I
_ CHLOROFORMNITROPHENOL REAGENT 4
Fig.
2.
Improved
!0.16!
SAMPLE
:1.60!
QUINOL-HzS04
;0.42:
AIR
I Il.29
! I
l1.M) I
I I
13.90 i
I
DILUENT WASTE
I
DILUENT
i I
I i3.90, I 1 I
I
I
I
I
WASTE
I
manifold.
reagent by way of a modified debubbler-cactus (Fig. 3). The made from the Technicon fittings “HO cactus connector” (part 0207-00) and “C4-T-connector” (part number 116-0202-04). It change from air to chloroform segmentation with a minimum and a consequent reduction of carry over.
l 2.4mm 0
Imm
Fig. 3. Debubbler-cactus.
device can be number 116allows for the of dead space
TABLE
I
CARRY-OVER ERAL
CHARACTERISTICS
RATES
OF
Sample
Wash
time
time
(s)
AND
PRECISION
OF
CONVENTIONAL
MANIFOLD
Rate (s)
of sampling
(samples/h)
Carry-over (% top
C.V.
% at mg/2
1
20
4.4
1.7
3.4
75
40
11.0
2.4
4.8
12
60
50
15.0
4.0
N.D.
150
1
32 mg/2
15
* Not
SFV-
std) 6.5
30
AT
SAMPLING
*
determined.
Results and discussion Carry-over becomes unacceptable in a continuous flow analyser when low samples can be so contaminated that re-analysis is necessary. It is important that in the analysis of urinary oestrogens in pregnancy low values should be accurately reported in order that possible dangers to the foetus be highlighted. The results obtained with a conventional system (Table I) suggest that at rates of sampling greater than 20 per hour carry-over would be too high to fulfill these conditions. Even at this low rate it was 4.4%. In contrast this figure was not surpassed by the improved system until a rate of 60 per hour had been exceeded, which represents a tripling of analysis rate with an actual improvement in carry-over (Table II). At rates greater than 67 per hour, with a variety of sample : wash ratios, carry-over became unacceptable. The precision of the method, especially in the important low value group, was not significantly impaired by the change in manifold design or increase in sampling rate up to 67 per hour. With ever increasing demands for urinary oestrogen analysis for the management of pregnancy, this modification will enable the extra workload to be assimilated by the chemical pathology laboratory with available staff and equipment. TABLE
II
CARRY-OVER RATES
OF
CHARACTERISTICS
Sample
Wash
time
time
(s)
AND
PRECISION
OF
IMPROVED
Rate (s)
of sampling
(samples/h)
Carry-over (W top
AT
SEVERAL
C.V.
(96) at
std) 6.5
mgl2
1
32
\l
2.0
40
cl
1.0
1.7
50
%.l
1.5
3.5
50
60
4.1
3.6
7.8
45
61
7.1
3.4
4.0
150
20
15
75
12
60
10 9
30
MANIFOLD
SAMPLING
2.0
8
40
75
15.2
10
40
72
25.0
1.4
N.D.
5
45
72
28.0
4.6
N.D.
--
11.6
mg/21
N.D.
___
__
Acknawledgements We wish to thank Dr, R.H. Wilkinson, in whose department carried out, and Mr. P. Tosh who made the debubblercactus.
this work was
References 1 Campbell, D.G. and Gardner, G. (1971) Clin, Chim. Acta 32,153 2 Fournier. A., Shields, T.W.. Neil, R.R.. Hayes, C.H. and Papineau-Coutre, G. (1966) Technicon Symp&urn, New York, N.Y. 3 Hainaworth. I.R. and Hall, P.E. (1971) Clin. Chim. Acta 35,201 4 Van Kessel. H., Satzinger. R., Schreurs, J. and Veertee& M. (1969) Ned. T. Verlosk. 69,Sl 5 tee, L. and Hiihnel. R. (1971) Clin. Chem. 17. 1204 6 Muir. C.G., Ua Connaill, D. and Ryan. M. (1969) Steroids 13,720 7 Muir, G.C. and Ryan, M. (1971) Ciin. Cbem. 17,1007 8 Ryan, M. and Gray, B.C. (1975) Clm. Chim. Acta 60,197-204 9 Strickler, MS.. Hoit, S.S., Acevedo, H.F., Saier, E. and Graver. R.C. (1967) Steroids 9.193
Amsterdam
- Printed
in The Netherlands
CCA 7100
AN IMPROVED MANIFOLD OESTROGEN ASSAY
R.A. MOORE, Nuffield
(Received
DESIGN FOR RAPID AUTOMATED
M. RYAN and W.A.F. PENFOLD
Department
September
of Clinical
Biochemistry,
Radcliffe
Infirmary,
Oxford
OX2 6HE
(U.K.)
24, 1975)
Summary
A modified “debubbler-cactus” provides for a change from air to chloroform segmentation in an automated fluorimetric oestrogen assay. The reduction in dead space allows the rate of sampling to be increased to 60 per hour with good precision and acceptable carry-over.
Introduction
In response to the increased demand for urinary oestrogen analysis as a monitor of foetal-placental well-being, a number of automated methods became available to cope with the load in the late 1960s. The fastest rates of analysis were of the order of 20 per hour (Fournier et al., 1966 [2] ; Strickler et al., 1967 [9] ; Van Kessel et al., 1969 [4] ; Muir et al., 1969 [6] and 1971 [7] ; Lee and Htihnel, 1971 [ 51, and Hainsworth and Hall, 1971 [3]), although Campbell and Gardner (1971) [l] proposed a method which yielded a rate of 30 per hour. This paper presents a modification in the manifold design of Ryan and Gray (1975) [8], which allows analysis rate to be increased to 60 per hour without loss of precision or increase in carry-over. Materials and methods
Reagents All reagents were A.R. grade and were purchased from BDH Chemicals, Poole, Dorset. Quinol-sulphuric acid: 15 g of quinol are shaken with 350 ml of distilled water, and 650 ml of cone HzS04 are added rapidly enough to cause boiling. When cooled the solution is made up to 1 litre with distilled water. Chloroform/nitrophenol: 20 g/l of 4-nitrophenol in chloroform.
2
Diluent: 3.75 g/l Brij 35 in distilled water for the conventional manifold, and 1.25 g/l Brij 35 in the improved system. These concentrations gave the best phase separation. Twenty-four hour urine samples were collected into bottles containing a small amount of thiomersal as preservative. The volurnc~ was recorded and where necessary the samples were diluted to 2 I with water. Ten ml aliquots were taken for pretreatment with sodium borohydride to eradicate glucose interference (Ryan and Gray, 1975 [ Sj ). Standard Autoanalyzer equipment (Technicon Instruments Corp., Tarrytown, N.Y. 10591) was used, with the exception of a Newton Sampler with variable sample and wash times (Burkard Scientific Sales Ltd., Woodcock Hill, Rickmansworth, Her&. WD3 1PJ) and a Fluorimeter Mk IV (Locarte Co., 8 Wendell Road, London, W12) equipped with a thallium light source. At various rates of sampling carry-over was determined by sampling a 10 mg/Z 1 standard immediately after a 40 mg/2 1 standard. The average increase in the low standard over three such cycles was expressed as a percentage of top standard. Precision was determined by repeated sampling of t,wo urines at levels (average) of 6.5 mg/2 1 and 32 mg/2 1. The precision of the method is expressed as coefficient of variation (C.V.).
The essential difference between the conventional manifold (Fig. 1) and the improved design (Fig. 2) is the direct injection of the reacted sample (which has passed through a heating bath and cooling coil) into the nitrophenol/chloroform
CONVENTIONAL
MANIFOLD HEATING
BATH
PUMP ml/min
To.
SAMPLE
! 1.601
QUINOL-H2S04
10.42: .
.
I
I
I Il.201
I
I
I t
Fig.
1. Conventional
manifold.
I I I
AIR
DILUENT
1
IMPROVED
MANIFOLD PUMP HEATING
ml/min
BATH
I
_ CHLOROFORMNITROPHENOL REAGENT 4
Fig.
2.
Improved
!0.16!
SAMPLE
:1.60!
QUINOL-HzS04
;0.42:
AIR
I Il.29
! I
l1.M) I
I I
13.90 i
I
DILUENT WASTE
I
DILUENT
i I
I i3.90, I 1 I
I
I
I
I
WASTE
I
manifold.
reagent by way of a modified debubbler-cactus (Fig. 3). The made from the Technicon fittings “HO cactus connector” (part 0207-00) and “C4-T-connector” (part number 116-0202-04). It change from air to chloroform segmentation with a minimum and a consequent reduction of carry over.
l 2.4mm 0
Imm
Fig. 3. Debubbler-cactus.
device can be number 116allows for the of dead space
TABLE
I
CARRY-OVER ERAL
CHARACTERISTICS
RATES
OF
Sample
Wash
time
time
(s)
AND
PRECISION
OF
CONVENTIONAL
MANIFOLD
Rate (s)
of sampling
(samples/h)
Carry-over (% top
C.V.
% at mg/2
1
20
4.4
1.7
3.4
75
40
11.0
2.4
4.8
12
60
50
15.0
4.0
N.D.
150
1
32 mg/2
15
* Not
SFV-
std) 6.5
30
AT
SAMPLING
*
determined.
Results and discussion Carry-over becomes unacceptable in a continuous flow analyser when low samples can be so contaminated that re-analysis is necessary. It is important that in the analysis of urinary oestrogens in pregnancy low values should be accurately reported in order that possible dangers to the foetus be highlighted. The results obtained with a conventional system (Table I) suggest that at rates of sampling greater than 20 per hour carry-over would be too high to fulfill these conditions. Even at this low rate it was 4.4%. In contrast this figure was not surpassed by the improved system until a rate of 60 per hour had been exceeded, which represents a tripling of analysis rate with an actual improvement in carry-over (Table II). At rates greater than 67 per hour, with a variety of sample : wash ratios, carry-over became unacceptable. The precision of the method, especially in the important low value group, was not significantly impaired by the change in manifold design or increase in sampling rate up to 67 per hour. With ever increasing demands for urinary oestrogen analysis for the management of pregnancy, this modification will enable the extra workload to be assimilated by the chemical pathology laboratory with available staff and equipment. TABLE
II
CARRY-OVER RATES
OF
CHARACTERISTICS
Sample
Wash
time
time
(s)
AND
PRECISION
OF
IMPROVED
Rate (s)
of sampling
(samples/h)
Carry-over (W top
AT
SEVERAL
C.V.
(96) at
std) 6.5
mgl2
1
32
\l
2.0
40
cl
1.0
1.7
50
%.l
1.5
3.5
50
60
4.1
3.6
7.8
45
61
7.1
3.4
4.0
150
20
15
75
12
60
10 9
30
MANIFOLD
SAMPLING
2.0
8
40
75
15.2
10
40
72
25.0
1.4
N.D.
5
45
72
28.0
4.6
N.D.
--
11.6
mg/21
N.D.
___
__
Acknawledgements We wish to thank Dr, R.H. Wilkinson, in whose department carried out, and Mr. P. Tosh who made the debubblercactus.
this work was
References 1 Campbell, D.G. and Gardner, G. (1971) Clin, Chim. Acta 32,153 2 Fournier. A., Shields, T.W.. Neil, R.R.. Hayes, C.H. and Papineau-Coutre, G. (1966) Technicon Symp&urn, New York, N.Y. 3 Hainaworth. I.R. and Hall, P.E. (1971) Clin. Chim. Acta 35,201 4 Van Kessel. H., Satzinger. R., Schreurs, J. and Veertee& M. (1969) Ned. T. Verlosk. 69,Sl 5 tee, L. and Hiihnel. R. (1971) Clin. Chem. 17. 1204 6 Muir. C.G., Ua Connaill, D. and Ryan. M. (1969) Steroids 13,720 7 Muir, G.C. and Ryan, M. (1971) Ciin. Cbem. 17,1007 8 Ryan, M. and Gray, B.C. (1975) Clm. Chim. Acta 60,197-204 9 Strickler, MS.. Hoit, S.S., Acevedo, H.F., Saier, E. and Graver. R.C. (1967) Steroids 9.193
