155
Clinica Chimica Acta, 64 (1975) 155-164 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7325
EVALUATION OF THE VITATRON ENZYME SYSTEM M.J. McQUEEN* Department (Received
AUTOMATIC
KINETIC
and J. KING
of Biochemistry,
Royal Infirmary, Glasgow, G4 OSF (U.K.)
April 19, 1975)
Introduction Enzyme analyses continue to constitute an increasing proportion of the workload of many clinical chemistry laboratories. In parallel with the recent improvements in methodologies there have been considerable improvements in instrumentation. The resulting proliferation of enzyme analysers can be confusing to a potential purchaser. Any mistakes in judgement will certainly prove to be expensive. There have been many fascinating advances introduced by the manufacturers of kinetic enzyme analysers but the weaknesses of the various systems have received considerably less publicity. The Vitatron Automatic Kinetic Enzyme System (AKES) was provided by the Scottish Home and Health Department (Equipment Research Committee) and was evaluated during the period March 1974 to June 1974. The scheme followed for the evaluation was obtained from reports compiled by working parties of the Laboratory Equipment and Methods Advisory Group [ 1,2]. System Description The system carries out the two basic processes of sample processing and data processing. Sample processing is performed by two dispensers and a diluter, all equipped with plug-in interchangeable Hamilton syringes and teflon tubing. The diluter employs adjustable syringes of 100 ~1 (sample) and 1000 ~1 (dilution) capacity and the dispenser used to initiate the reaction is equipped with a 100 ~1 adjustable syringe. A chain made up from ten link sections, each link containing 10 sample places and being numbered and digitally coded, transports through the system
* Present Canada.
address:
Department
of Clinical
Chemistry,
Camp
Hill Hospital.
Halifax,
Nova
Scotia,
156
disposable plastic sample vials containing up to 600 pi of sample. The sample is removed from each vial by the probe needle piercing the sealed plastic top and the sample is washed out of the probe by the buffer or substrate contained in the dilution system. The sample is delivered via the probe into a glass measuring cuvette which has a maximum reaction volume of 800 ~1 and a minimum of 500 pl. The cuvettes are contained in a rotating, thermostatted 18 place disc. From sample point to measuring point consists of 14 steps and the minimum incubation time for the first sample is 4 min and 40 s. All other samples have a variable incubation time depending upon how long the preceeding reaction(s) takes to achieve acceptable linearity. Heating and cooling is by means of a Peltier element and the user can select a temperature of 25, 30, 35 or 37°C. Each cuvette is cleaned after it moves from the measuring position. Water and a suitable detergent are delivered by the second dispenser which contains an adjustable 1000 ~1 syringe. Discarded sample, reagents and water are removed via a suction system. The photometer is equipped with interference filters (lo-nm bandwidth) and two light sources, a mercury lamp for measurements at 334 and 405 nm, and a quartz iodine lamp for use at 340 nm. Data processing is carried out by a BCD sample number reader which ensures that samples and results are correctly related, a Reaction Rate Computer and a digital printer. If the sample identification lamp is defective audio-visual alarms warn the user and the whole system ceases to operate. Similar alarms also operate if there is no starting reagent or buffer/substrate, if the sample chain becomes blocked, when the end of the sample chain is reached, or when the waste reservoir is full. The reaction rate computer (RRC) calculates the enzyme activity (U/l) by multiplying AA/min and the factor (O-10 000) for each enzyme assay. If AA/min is GO.002 or 20.400 the result from such a specimen is rejected by the computer and an error code is printed. The RRC also checks and calculates data relating to the initial absorbance and linearity. The initial absorbance, which represents the sum of the absorbance of the serum and the absorbance of the reaction mixture, usually lies between a certain minimum and maximum value. If it is too low or too high the RRC is programmed to cause the printer to indicate an error code number for each of these instances and again no result is printed. If all these criteria are met the reaction rate is followed continuously by the computer. Prior to the sample being measured a linearity deviation limit of lo%, 5%, 2% or 1% is selected. If the reaction rate lies within the selected limit for a period of 20 s the change of absorbance is multiplied by the appropriate factor and is printed out by the digital printer. If the reaction rate is outside the selected linearity limit the computer follows the reaction for another 20 s and if it is within the selected Iimits the result is printed. If it is not, then another 40 s of measurement is done and so on if required to a maximum of 2 min 46 s. There is, therefore, a minimum measuring time of 20 s and a maximum of 2 min 46 s, The RRC calculating principle is a quadratic integration method applied to the various measuring times of 20, 42, 84 or 166 s.
157
Capital cost and running costs At the time the cost of the complete system was the same as the LKB 8600 reaction rate analyser, calculator with mosaic printer and sampler (Hook and Tucker K40) a system performing the same function. We found that there were no significant differences in technician time involved in operating both these systems. The measuring cuvettes are washed and re-used in the AKES whereas they are disposable for the LKB system. Thus there would appear to be a significant saving in the cost of disposable items with the Vitatron system. With the AKES however there is the possible expense of replacing damaged glass cuvettes. Only a prolonged assessment under routine conditions will disclose how often this is likely to occur. A further saving in running costs may be in reagents since the AKES uses about half the volumes usually, but not necessarily, employed in the LKB system. The last however is also somewhat dependent upon the number of assays performed, storage life and wastage in priming. Instrument
dependability
and servicing
During the period of this assessment the instrument was operational performing analyses for 468 hours. Down time resulted in the loss of 153 working hours, which represented 24.6% of the available working time. A total of 46 800 analyses were performed. There were several faults which led to this time loss. The most substantial was a faulty RRC which required to be replaced by the manufacturers. The light source was also troublesome and the machine was using its third lamp by the end of the assessment. One cuvette was damaged and the carousel replaced. Subsequently a second cuvette was also suspected of damage and was replaced also. However, the manufacturers claim that this is not likely to be a recurring problem and it is now possible to have a damaged cuvette removed and replaced by a service engineer rather than replacing the whole ring system. An insulation problem with the Peltier element resulted in a small modification to the instrument. The most consistent faults developed in the alarm systems. They were a source of alarm, not in serving their intended function of indicating system faults, but in developing faults themselves. A series of faulty micro-switches set off the alarm systems and thus, as intentioned, inactivated the entire system. We understand that as a result of this experience Vitatron have largely overcome this problem by means of tighter quality control. Since there was no service engineer resident in Scotland, this resulted in substantial delays before the faults could be rectified*. We could not fault the ability or enthusiasm of the service engineer who assisted us, but if the equipment had been providing a routine service function these delays would have been unacceptable.
* There
is now
a resident
Service
Engineer
in Scotland.
158 TABLE Precision
I and accuracy
of the Hamilton
syringes used in the dispensing
and diluting modules. _.__~
Syringes
set volume
(fil)
(/.ll)
Measured
volume
(~1) 01 = 20)
_....~__~ C.V. (sr,)
1000
1000 500 250 100
997.2 497.3 247.3 97.4
0.031 0.057 0.072 0.222
100
100 20
101.8 20.8
0.133 0.577 ~_~ _..~_.
.~~_~ -__
Dispensers
_
~
_~~..~~
-
and diluters
There are two Hamilton syringes, 100 ,ul and 1000 ~1, used in the dispensing and diluting modules. The accuracy and precision of these units was checked with twenty consecutive weighings of water dispended by the pumps. Table I gives details of the set and measured volumes obtained with each syringe. The coefficient of variation deteriorates as the lower settings are used with each pump. With the 1000 ,ul syringe the 100 ~1 setting is, however, rather an academic exercise, as a minimum reaction volume of 500 ,ul is needed and a setting less than 400 ,ul is not going to be required. With the 100 (~1syringe a 20 (~1setting produced a C.V. of 0.58%. If sample volumes of 20 ~1 or less were being used regularly, it would be better to substitute a smaller syringe in the place of the 100 ~1 one. Temperature
control
A temperature and the time taken TABLE
probe was inserted in the liquid in the measuring cuvette for heating and cooling to the selected temperatures was
II
Heating and cooling of the reaction mixture (ambient temperature 24%). the light switched off and the temperature that achieved at this time. further required before the steady temperature was achieved. Cooling
Heating ______~~~__. 25’C
25OC
3o”c
35Oc
37Oc
_
3.8 min 25.8’C 1.7 min 6.7 min 25.8”C 1.8 min 8.0 min 25.7’C 2.3 min
The upper time is that at which The lower time interval is that
~_____ ~_.
___..-. 30°c
35Oc
37Oc
2.8 min 28.8OC 1.7 min -
6.3 min 34.2OC 1.5 min 3.8 min 34.1°C 2.5 min
7.0 min 36.2OC 1.8 min 5.2 min 36.1°C 1.5 min 2.0 min 36.2OC 1.5 min -
3.7 min 31.1°C 2.2 min 4.7 min 31.2’C 2.0 min
2.8 min 35.6OC 2.0 min
159
measured (Table II). The results in this table can be understood by looking first at the heating results. On increasing the temperature from 25°C to 30°C the light switched off after 2.8 min when the temperature was at 28.8”C. A further 1.7 min passed before the contents of the cuvette attained a steady temperature. The light is therefore misleading, but it is not of practical significance as the time involved, 1.7-2.5 mm, is less than the time it takes for the plastic sample cuvette to reach the sampling position. On cooling it took 1.7 to 2.3 min longer for the sample cuvettes to lose heat after the temperature control light went out. Assessment
of accuracy
and precision
Within batch precision at 25°C and 37°C using the Dade UV-10 CPK one step procedure is given in Table III. Thirty 25-/J samples of pooled human serum and also thirty samples of Dade CK control were measured at 334 nm. The C.V. improved with the increased activity which resulted from raising the temperature from 25°C to 37°C. The effect of sample volume on precision is detailed in Table IV, the enzyme activity being measured at 37°C at a wavelength of 334 nm. When the sample volume was reduced from 25 ~1 to 10 1.11 the CK control (Dade) which previously had a mean activity of 364 U/l, showed a deterioration in the C.V. from 1.94% to 3.65%. The practical effect of the different linearity setting is also illustrated when the enzyme activity is measured using the 10-/-d samples. When 10% linearity was selected a C.V. of 3.55% was found, yet this was reduced to 1.88% when a 5% linearity deviation was selected. To show the effect of the linearity setting on precision and on throughput, thirty 25-/J samples of pooled human sera were measured at each of the available linearity settings. The improvements in C.V. with the lower linearity settings and the slower throughput which is the price of this greater precision are shown in Table V and were not as pronounced as expected. The results suggest that in routine use the 10% setting is acceptable for this creatine kinase assay procedure. Several enzymes were measured in addition to creatine kinase. Lactate dehydrogenase (LDH-Boehringer) assays at 25°C and 37°C using pooled sera and several quality control materials gave acceptable coefficients of variation (Table VI). The pooled human sera with an activity of 1052 U/l at 25°C was not measureable at 37°C and the reaction rate computer indicated via, the printer that the reaction rate was too high because AA/min aO.4. Alpha-hydroxybutyrate dehydrogenase activities were measured using International Diagnostic Aids (IDA) reagents and the results are given in Table VII. Gamma-glutamyl transpeptidase was measured at 25°C and 37°C using a Boehringer test kit. Good precision was obtained for all specimens measured at both temperatures (Table VIII), but precision at 25°C was superior to that at 37°C. Throughput at 25°C was 110 samples per hour compared with 98 samples per hour at 37°C. This suggests the possibility that since the reaction rate computer was set at 5% deviation from linearity, the higher temperature resulted in less linear tracings. Sub-optimal reaction conditions for the higher temperature could have been the cause for the poorer linearity.
160 TABLE
III
Within
batch
deviation
precision
Human pool Dade CK control
Creatine 334 nm. Sample
and 37’C
using the Dade UV-10
CK one step procedure.
25°C
Sample
TABLE
at 25’C
A 5% linearity
was selected. 37°C _.
s- (U/l)
s
25 142
2.33 4.69
(U/l)
C.V. (S)
x
9.4 3.3
60 364
.~~
(U/l)
s (U/l)
C.V. (%)
3.38 7.09
5.6 1.94
IV kinase
activity
sample
volumes
of CK control
material
measured
at 37’C
x- (U/l)
s (U/I)
C.V. (%)
Linearity
25 IO
364 361
7.09 14.17
1.94 3.65
10
10 10
359 357
11.85 6.73
3.55 1.88
10 5
TABLE
(cll)
with different
setting (W)
10 5 2
TABLE
“0
V
The effect of the linearity settings on precision and throughput sera. Activities were measured at 334 nm and at 37’C. Linearity
and
of CK measurements
of pooled
%m(U/l)
s (U/l)
C.V. (%)
Throughput
330 331 327
7.0 6.73 6.4
2.12 2.04 1.96
80 75 71
(samples
human
/h)
VI
LDH activities men?..
(Boehringer
Sample
reagents)
and 37’C
of pooled
25OC x
Human pool Human pool Boehringer Precinorm Boehringer Precipath Dade Chemonitor 1 Dade Enzatrol Dade Chemonitor II
at 25’C
(U/I)
310 1052 336 712 203 1050 961
human
sera and wahtv
control
SPeci-
37Oc s (U/I)
C.V. (%)
14.5 15.44 11.31 14.01 7.2 26.8 16.1
4.69 1.47 3.37 1.97 3.55 2.55 1.68
x
(U/I)
_ 649
1384 451 1918 1735
s
(U/l)
10.58 25.7 16.46 8.63 16.7 16.47
C.V. (%)
-
1.76
3.96 1.19 1.91 0.87 0.95
161
TABLE
VII
n-HBD
activities
(IDA
reagents)
Sample
at 25OC
and
37”C
of
pooled
human
Human
pool
Human
pool
and
quality
control
specimens.
37Oc
25OC
Y
sera
(U/l)
s (U/l)
C.V.
4.53
93 218
x
(%)
s (U/l)
(U/l)
4.90
128
6.55
5.12
12.80
5.88
295
5.73
1.94
Boehringer
Precinorm
82
6.14
7.47
126
3.66
2.89
Boehringer
Precipath
197
6.87
3.49
283
8.96
3.17
63
6.96
11.12
201
10.05
5.01
266
lo.85
5.21
275
10.48
3.81
4.51
222
9.23
4.15
Dade
Chemonitor
I
Dade
Chemonitor
II
Dade
Enzatrol
208
BDH
Scronorm
178
TABLE
3.74
4.32
5.44
2.05
VIII
Gamma-glutamyl and
8.014
87
(%)
C.V.
Chemonitor
transpeptidase I quality
Sample
activities
control
(Boehringer
reagents)
at 25’C
25’C
x
and
37’C
of
pooled
human
sera
serum.
370.4. A subjective assessment of the instrument is that it is easy to use but requires more careful handling by technicians than they have become ac* The recently introduced computer for substrate estimations has a rejection limit of 0.3 AA/min.
164
customed to giving to other automated equipment. It is versatile and could make a substantial contribution to handling the variety of enzyme requests being faced by many routine clinical chemistry laboratories. Acknowledgments We gratefully acknowledge the assistance and encouragement of Mr Eric Leask of the Scottish Home and Health Department at all stages of this evaluation. References 1 Broughton, P.M.G., Buttolph, M.A., Gowenlock, A.H., Neil& D.W. and Skentelberry. R.G. (19691 J. C&I. Pathol. 22, 278-284 2 Broughton, P.M.G., Gowenlock, A.H., McCormack, J.J. and NeiU, D.W. (1974) Ann. Clin. Bioehem. 11,207-218
Clinica Chimica Acta, 64 (1975) 155-164 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7325
EVALUATION OF THE VITATRON ENZYME SYSTEM M.J. McQUEEN* Department (Received
AUTOMATIC
KINETIC
and J. KING
of Biochemistry,
Royal Infirmary, Glasgow, G4 OSF (U.K.)
April 19, 1975)
Introduction Enzyme analyses continue to constitute an increasing proportion of the workload of many clinical chemistry laboratories. In parallel with the recent improvements in methodologies there have been considerable improvements in instrumentation. The resulting proliferation of enzyme analysers can be confusing to a potential purchaser. Any mistakes in judgement will certainly prove to be expensive. There have been many fascinating advances introduced by the manufacturers of kinetic enzyme analysers but the weaknesses of the various systems have received considerably less publicity. The Vitatron Automatic Kinetic Enzyme System (AKES) was provided by the Scottish Home and Health Department (Equipment Research Committee) and was evaluated during the period March 1974 to June 1974. The scheme followed for the evaluation was obtained from reports compiled by working parties of the Laboratory Equipment and Methods Advisory Group [ 1,2]. System Description The system carries out the two basic processes of sample processing and data processing. Sample processing is performed by two dispensers and a diluter, all equipped with plug-in interchangeable Hamilton syringes and teflon tubing. The diluter employs adjustable syringes of 100 ~1 (sample) and 1000 ~1 (dilution) capacity and the dispenser used to initiate the reaction is equipped with a 100 ~1 adjustable syringe. A chain made up from ten link sections, each link containing 10 sample places and being numbered and digitally coded, transports through the system
* Present Canada.
address:
Department
of Clinical
Chemistry,
Camp
Hill Hospital.
Halifax,
Nova
Scotia,
156
disposable plastic sample vials containing up to 600 pi of sample. The sample is removed from each vial by the probe needle piercing the sealed plastic top and the sample is washed out of the probe by the buffer or substrate contained in the dilution system. The sample is delivered via the probe into a glass measuring cuvette which has a maximum reaction volume of 800 ~1 and a minimum of 500 pl. The cuvettes are contained in a rotating, thermostatted 18 place disc. From sample point to measuring point consists of 14 steps and the minimum incubation time for the first sample is 4 min and 40 s. All other samples have a variable incubation time depending upon how long the preceeding reaction(s) takes to achieve acceptable linearity. Heating and cooling is by means of a Peltier element and the user can select a temperature of 25, 30, 35 or 37°C. Each cuvette is cleaned after it moves from the measuring position. Water and a suitable detergent are delivered by the second dispenser which contains an adjustable 1000 ~1 syringe. Discarded sample, reagents and water are removed via a suction system. The photometer is equipped with interference filters (lo-nm bandwidth) and two light sources, a mercury lamp for measurements at 334 and 405 nm, and a quartz iodine lamp for use at 340 nm. Data processing is carried out by a BCD sample number reader which ensures that samples and results are correctly related, a Reaction Rate Computer and a digital printer. If the sample identification lamp is defective audio-visual alarms warn the user and the whole system ceases to operate. Similar alarms also operate if there is no starting reagent or buffer/substrate, if the sample chain becomes blocked, when the end of the sample chain is reached, or when the waste reservoir is full. The reaction rate computer (RRC) calculates the enzyme activity (U/l) by multiplying AA/min and the factor (O-10 000) for each enzyme assay. If AA/min is GO.002 or 20.400 the result from such a specimen is rejected by the computer and an error code is printed. The RRC also checks and calculates data relating to the initial absorbance and linearity. The initial absorbance, which represents the sum of the absorbance of the serum and the absorbance of the reaction mixture, usually lies between a certain minimum and maximum value. If it is too low or too high the RRC is programmed to cause the printer to indicate an error code number for each of these instances and again no result is printed. If all these criteria are met the reaction rate is followed continuously by the computer. Prior to the sample being measured a linearity deviation limit of lo%, 5%, 2% or 1% is selected. If the reaction rate lies within the selected limit for a period of 20 s the change of absorbance is multiplied by the appropriate factor and is printed out by the digital printer. If the reaction rate is outside the selected linearity limit the computer follows the reaction for another 20 s and if it is within the selected Iimits the result is printed. If it is not, then another 40 s of measurement is done and so on if required to a maximum of 2 min 46 s. There is, therefore, a minimum measuring time of 20 s and a maximum of 2 min 46 s, The RRC calculating principle is a quadratic integration method applied to the various measuring times of 20, 42, 84 or 166 s.
157
Capital cost and running costs At the time the cost of the complete system was the same as the LKB 8600 reaction rate analyser, calculator with mosaic printer and sampler (Hook and Tucker K40) a system performing the same function. We found that there were no significant differences in technician time involved in operating both these systems. The measuring cuvettes are washed and re-used in the AKES whereas they are disposable for the LKB system. Thus there would appear to be a significant saving in the cost of disposable items with the Vitatron system. With the AKES however there is the possible expense of replacing damaged glass cuvettes. Only a prolonged assessment under routine conditions will disclose how often this is likely to occur. A further saving in running costs may be in reagents since the AKES uses about half the volumes usually, but not necessarily, employed in the LKB system. The last however is also somewhat dependent upon the number of assays performed, storage life and wastage in priming. Instrument
dependability
and servicing
During the period of this assessment the instrument was operational performing analyses for 468 hours. Down time resulted in the loss of 153 working hours, which represented 24.6% of the available working time. A total of 46 800 analyses were performed. There were several faults which led to this time loss. The most substantial was a faulty RRC which required to be replaced by the manufacturers. The light source was also troublesome and the machine was using its third lamp by the end of the assessment. One cuvette was damaged and the carousel replaced. Subsequently a second cuvette was also suspected of damage and was replaced also. However, the manufacturers claim that this is not likely to be a recurring problem and it is now possible to have a damaged cuvette removed and replaced by a service engineer rather than replacing the whole ring system. An insulation problem with the Peltier element resulted in a small modification to the instrument. The most consistent faults developed in the alarm systems. They were a source of alarm, not in serving their intended function of indicating system faults, but in developing faults themselves. A series of faulty micro-switches set off the alarm systems and thus, as intentioned, inactivated the entire system. We understand that as a result of this experience Vitatron have largely overcome this problem by means of tighter quality control. Since there was no service engineer resident in Scotland, this resulted in substantial delays before the faults could be rectified*. We could not fault the ability or enthusiasm of the service engineer who assisted us, but if the equipment had been providing a routine service function these delays would have been unacceptable.
* There
is now
a resident
Service
Engineer
in Scotland.
158 TABLE Precision
I and accuracy
of the Hamilton
syringes used in the dispensing
and diluting modules. _.__~
Syringes
set volume
(fil)
(/.ll)
Measured
volume
(~1) 01 = 20)
_....~__~ C.V. (sr,)
1000
1000 500 250 100
997.2 497.3 247.3 97.4
0.031 0.057 0.072 0.222
100
100 20
101.8 20.8
0.133 0.577 ~_~ _..~_.
.~~_~ -__
Dispensers
_
~
_~~..~~
-
and diluters
There are two Hamilton syringes, 100 ,ul and 1000 ~1, used in the dispensing and diluting modules. The accuracy and precision of these units was checked with twenty consecutive weighings of water dispended by the pumps. Table I gives details of the set and measured volumes obtained with each syringe. The coefficient of variation deteriorates as the lower settings are used with each pump. With the 1000 ,ul syringe the 100 ~1 setting is, however, rather an academic exercise, as a minimum reaction volume of 500 ,ul is needed and a setting less than 400 ,ul is not going to be required. With the 100 (~1syringe a 20 (~1setting produced a C.V. of 0.58%. If sample volumes of 20 ~1 or less were being used regularly, it would be better to substitute a smaller syringe in the place of the 100 ~1 one. Temperature
control
A temperature and the time taken TABLE
probe was inserted in the liquid in the measuring cuvette for heating and cooling to the selected temperatures was
II
Heating and cooling of the reaction mixture (ambient temperature 24%). the light switched off and the temperature that achieved at this time. further required before the steady temperature was achieved. Cooling
Heating ______~~~__. 25’C
25OC
3o”c
35Oc
37Oc
_
3.8 min 25.8’C 1.7 min 6.7 min 25.8”C 1.8 min 8.0 min 25.7’C 2.3 min
The upper time is that at which The lower time interval is that
~_____ ~_.
___..-. 30°c
35Oc
37Oc
2.8 min 28.8OC 1.7 min -
6.3 min 34.2OC 1.5 min 3.8 min 34.1°C 2.5 min
7.0 min 36.2OC 1.8 min 5.2 min 36.1°C 1.5 min 2.0 min 36.2OC 1.5 min -
3.7 min 31.1°C 2.2 min 4.7 min 31.2’C 2.0 min
2.8 min 35.6OC 2.0 min
159
measured (Table II). The results in this table can be understood by looking first at the heating results. On increasing the temperature from 25°C to 30°C the light switched off after 2.8 min when the temperature was at 28.8”C. A further 1.7 min passed before the contents of the cuvette attained a steady temperature. The light is therefore misleading, but it is not of practical significance as the time involved, 1.7-2.5 mm, is less than the time it takes for the plastic sample cuvette to reach the sampling position. On cooling it took 1.7 to 2.3 min longer for the sample cuvettes to lose heat after the temperature control light went out. Assessment
of accuracy
and precision
Within batch precision at 25°C and 37°C using the Dade UV-10 CPK one step procedure is given in Table III. Thirty 25-/J samples of pooled human serum and also thirty samples of Dade CK control were measured at 334 nm. The C.V. improved with the increased activity which resulted from raising the temperature from 25°C to 37°C. The effect of sample volume on precision is detailed in Table IV, the enzyme activity being measured at 37°C at a wavelength of 334 nm. When the sample volume was reduced from 25 ~1 to 10 1.11 the CK control (Dade) which previously had a mean activity of 364 U/l, showed a deterioration in the C.V. from 1.94% to 3.65%. The practical effect of the different linearity setting is also illustrated when the enzyme activity is measured using the 10-/-d samples. When 10% linearity was selected a C.V. of 3.55% was found, yet this was reduced to 1.88% when a 5% linearity deviation was selected. To show the effect of the linearity setting on precision and on throughput, thirty 25-/J samples of pooled human sera were measured at each of the available linearity settings. The improvements in C.V. with the lower linearity settings and the slower throughput which is the price of this greater precision are shown in Table V and were not as pronounced as expected. The results suggest that in routine use the 10% setting is acceptable for this creatine kinase assay procedure. Several enzymes were measured in addition to creatine kinase. Lactate dehydrogenase (LDH-Boehringer) assays at 25°C and 37°C using pooled sera and several quality control materials gave acceptable coefficients of variation (Table VI). The pooled human sera with an activity of 1052 U/l at 25°C was not measureable at 37°C and the reaction rate computer indicated via, the printer that the reaction rate was too high because AA/min aO.4. Alpha-hydroxybutyrate dehydrogenase activities were measured using International Diagnostic Aids (IDA) reagents and the results are given in Table VII. Gamma-glutamyl transpeptidase was measured at 25°C and 37°C using a Boehringer test kit. Good precision was obtained for all specimens measured at both temperatures (Table VIII), but precision at 25°C was superior to that at 37°C. Throughput at 25°C was 110 samples per hour compared with 98 samples per hour at 37°C. This suggests the possibility that since the reaction rate computer was set at 5% deviation from linearity, the higher temperature resulted in less linear tracings. Sub-optimal reaction conditions for the higher temperature could have been the cause for the poorer linearity.
160 TABLE
III
Within
batch
deviation
precision
Human pool Dade CK control
Creatine 334 nm. Sample
and 37’C
using the Dade UV-10
CK one step procedure.
25°C
Sample
TABLE
at 25’C
A 5% linearity
was selected. 37°C _.
s- (U/l)
s
25 142
2.33 4.69
(U/l)
C.V. (S)
x
9.4 3.3
60 364
.~~
(U/l)
s (U/l)
C.V. (%)
3.38 7.09
5.6 1.94
IV kinase
activity
sample
volumes
of CK control
material
measured
at 37’C
x- (U/l)
s (U/I)
C.V. (%)
Linearity
25 IO
364 361
7.09 14.17
1.94 3.65
10
10 10
359 357
11.85 6.73
3.55 1.88
10 5
TABLE
(cll)
with different
setting (W)
10 5 2
TABLE
“0
V
The effect of the linearity settings on precision and throughput sera. Activities were measured at 334 nm and at 37’C. Linearity
and
of CK measurements
of pooled
%m(U/l)
s (U/l)
C.V. (%)
Throughput
330 331 327
7.0 6.73 6.4
2.12 2.04 1.96
80 75 71
(samples
human
/h)
VI
LDH activities men?..
(Boehringer
Sample
reagents)
and 37’C
of pooled
25OC x
Human pool Human pool Boehringer Precinorm Boehringer Precipath Dade Chemonitor 1 Dade Enzatrol Dade Chemonitor II
at 25’C
(U/I)
310 1052 336 712 203 1050 961
human
sera and wahtv
control
SPeci-
37Oc s (U/I)
C.V. (%)
14.5 15.44 11.31 14.01 7.2 26.8 16.1
4.69 1.47 3.37 1.97 3.55 2.55 1.68
x
(U/I)
_ 649
1384 451 1918 1735
s
(U/l)
10.58 25.7 16.46 8.63 16.7 16.47
C.V. (%)
-
1.76
3.96 1.19 1.91 0.87 0.95
161
TABLE
VII
n-HBD
activities
(IDA
reagents)
Sample
at 25OC
and
37”C
of
pooled
human
Human
pool
Human
pool
and
quality
control
specimens.
37Oc
25OC
Y
sera
(U/l)
s (U/l)
C.V.
4.53
93 218
x
(%)
s (U/l)
(U/l)
4.90
128
6.55
5.12
12.80
5.88
295
5.73
1.94
Boehringer
Precinorm
82
6.14
7.47
126
3.66
2.89
Boehringer
Precipath
197
6.87
3.49
283
8.96
3.17
63
6.96
11.12
201
10.05
5.01
266
lo.85
5.21
275
10.48
3.81
4.51
222
9.23
4.15
Dade
Chemonitor
I
Dade
Chemonitor
II
Dade
Enzatrol
208
BDH
Scronorm
178
TABLE
3.74
4.32
5.44
2.05
VIII
Gamma-glutamyl and
8.014
87
(%)
C.V.
Chemonitor
transpeptidase I quality
Sample
activities
control
(Boehringer
reagents)
at 25’C
25’C
x
and
37’C
of
pooled
human
sera
serum.
370.4. A subjective assessment of the instrument is that it is easy to use but requires more careful handling by technicians than they have become ac* The recently introduced computer for substrate estimations has a rejection limit of 0.3 AA/min.
164
customed to giving to other automated equipment. It is versatile and could make a substantial contribution to handling the variety of enzyme requests being faced by many routine clinical chemistry laboratories. Acknowledgments We gratefully acknowledge the assistance and encouragement of Mr Eric Leask of the Scottish Home and Health Department at all stages of this evaluation. References 1 Broughton, P.M.G., Buttolph, M.A., Gowenlock, A.H., Neil& D.W. and Skentelberry. R.G. (19691 J. C&I. Pathol. 22, 278-284 2 Broughton, P.M.G., Gowenlock, A.H., McCormack, J.J. and NeiU, D.W. (1974) Ann. Clin. Bioehem. 11,207-218
