Journal of Automatic Chemistry Vol. 10, No. 4 (October-December 1988), pp. 181-183
Recommendations for definition and
determination of carry-over effects
R. Haeckel
Zentralkrankenhaus, Inst. ffir Laboratoriumsmedzin, D2800 Bremen 1,
FR Germany
Thefollowing concepts have been elaborated by the Commission on
Automation and New Technologies ofthe International Union of
Pure and Applied Chemistry [1].
Definitions
The term ’carry-over’ is commonly used to describe a
process by which materials are carried into a reaction
mixture to which they do not belong. These materials can
be either parts of a specimen or reagents, including
diluent or wash solution. In such cases carry-over means
transfer of material (specimen or reagents) from one
container or from one reaction mixture to another; it can
be either unidirectional or bidirectional in a series of
specimens or assays.
Classification
Carry-over can be classified according to either the
material which is carried over, or to the site where the
carry-over occurs, or to its dependency on the sample (see
table 1).
Table 1. Classification ofcarry-over effects.
1. According to the site where it occurs: carry-over in
specimen cup, sample probe, reagent probe, reaction
system, signal detection system, wash station.
2. According to the material which is carried over:
carry-over ofspecimen, diluent, reagent, reaction mixture,
wash solution.
3. According to the sample dependency.
3.1. Specimen-dependent carry-over.
3.2. Specimen-independent carry-over.
Current concepts for the determination ofcarry-over
The current concept ofcarry-over implies the transfer ofa
part ofone reaction mixture to another. It is measured in
terms of volume, mass or concentration fraction, either
determined independently from the analytical procedure
by using dye solutions or radioactive isotopes or by one of
the analytical procedures applied.
In analytical chemistry, the error produced by carry-over
within a particular procedure is of interest. It is dependent
on the method, including all reagents applied.
Therefore, carry-over effects, rather than carry-over
itself, need to be determined. For this purpose, a sequence
ofspecimens, usually with high (a) and low (b) concentrations
of the analyte to be measured are processed" for
example a, a2, bl, b2, b (see figure 1).
SIGNAL TO BE DETECTED
Figure 1.
h=, -b
h’ b b
b
h’ (.) h’ 100
(eq. I)
(eq. II)
a, a b, b2 b SEQUENCE OF SAMPLES
The carry-over effect (q) can be calculated using either
equation [2]
bl bq
a2 ba
(1)
or equation 2 [3]
q=a2-bl (2)
q may also be called the ’carry-over ratio’, and is
sometimes expressed as ’percentage value’ (Q 100 q).
It implies that the influence of carry-over effects on the
analytical results is inversely proportional to the
concentration difference ofthe component to be detected
by the analytical procedure in the two specimens. The
last assumption is probably met in many cases [2],
however, does not apply for pH/blood gas analysers and
not for some selective analysers where complicated
carry-over phenomena may occur from reagents to
reaction mixtures.
Another disadvantage ofcalculating the carry-over effect
(q) is that it does not indicate directly to what extent the
resuts may be erroneously influenced. A carry-over ratio
of q 0"01 (Q 1%) can lead to clinically misleading
results in one case whereas a q 0.1 (Q 10%) value
may be irrelevant in another case. This shall be demonstrated
by two examples which are well known to clinical
chemists.
In the first example q 0"01 and the ALT value following
a specimen with 2000 U/I will be 40 U/! instead of20 U/I.
in the second example q 10 times higher (q 0.1) and the
chloride concentration following a specimen with 140
181
R. Haeckel Recommendations for definition and determination of carry-over effects
mmol/1 will be 86 mmol/1 instead of 80 mmol/l. In one
situation the carry-over effect leads erroneously to a
pathological value with clinical relevance, whereas in the
other example the carry-over is clinically unimportant
although carry-over is 10 times higher.
Proposed concept for determining carry-over effects
Since the carry-over in an analytical system depends
partly on the reagents, carry-over effects in a spectrometer
(chosen as an example) should be determined
with the reagents used in the cuvette or flow cell of a
particular spectrometer rather than with dye solutions.
These effects should be expressed in units (for example
mol/1 or U/l) which are used to present the results and not
as quantity fractions. As already mentioned, carry-over
effects can be either specimen related or specimen
independent.
Specimen-dependent carry-over
Specimen-dependent carry-over either may occur with
one method using a constant sample volume or in
multitest analysers with sample volumes varying from
method to method. Only the first case is considered; the
second case is explained in reference [1].
Where the determination of a specimen with a high
concentration ofanalyte interferes with that ofa specimen
with a low concentration (or vice versa), a sequence ofat
least two successive aliquots of a specimen with a high
value (a), followed by at least three successive aliquots of
one with a low value (b), should be used to determine the
carry-over effect.
The concentration of (b) should be chosen to be close to
the most relevant decision level, which in many cases is
the upper limit of the reference interval (for example
20-30 U/1 in the case of aminotransferase catalytic
activity).
The concentration of (a) should represent the extreme
values which may occur (for example 1000-2000 U/1 for
aminotransferase activity concentration or 50 mmol/1 for
glucose concentration). The volume in the container from
which the sample is taken must be defined (usually it
should be filled to two-thirds of its maximum capacity).
This experiment should be repeated 10 times [5]. For the
comparison of the paired values bl (bl) and b3 (b3) the
Wilcoxon signed rank test is recommended. If a highly
significant (for example 0 -< 1%) difference is detected,
then the mean carry-over effect (h) is calculated see
(figure 1)"
h bl- ba (3)
The carry-over effect may also be expressed as a fraction
of ba or as percentage of the value ba.
When the carry-over effect is not highly significant, the
procedure can be classified as carry-over safe in an
interval between the quantity measured in specimen b
(lower limit) and the quantity measured in specimen a
(upper limit). After a carry-over effect has been detected,
it is common practice to repeat a test when the difference
between its result and the previous one suggests that a
significant error has occured due to carry-over effects.
Which difference can just be tolerated (that means the
carry-over safe interval) is usually estimated by intuition.
However, it can also be determined experimentally. For
this purpose, the experiment reported above must be
repeated with lower (a)-value(s) which are close to the
upper limit of the linear range. Ifa significant carry-over
effect cannot be detected, the upper limit ofthe carry-over
safe interval is assumed to be identical with the upper
limit ofthe linearity interval. Ifthe carry-over effect is still
significant, then the concentration at the upper limit of
the carry-over safe interval can be estimated. It shall be
assumed that
bl- ba -< b. + 2s
This means that the carry-over effect h b b3 should be
smaller than twice the standard deviation obtained with
this particular method. Furthermore it is assumed that
the carry-over effect increases linearily with the difference
of quantities measured in specimens a and b. The
carry-over safe interval can then be calculated by the
equation
2s(1 +q)
as= +ba (5)q
where s is the standard deviation calculated from the 10
b-values determined in the previous carry-over experiment;
q is calculated similarly to equation I from the
results of the previous carry-over experiment (the a value
is close to the upper limit of the linear range).
After the carry-over safe interval has been determined, all
results close to the reference range of the particular
analytical procedure should be regarded as falsely
increased if they follow a result above the carry-over safe
interval. This interval is not valid for results which are far
below the b-value chosen for the calculation of the
carry-over effect. In some cases the carry-over safe
interval may be relatively small and too many analyses
have to be repeated. Then, each result can be corrected
by using a factor q calculated according to either equation
I or II. However, such a situation should be avoided.
Specimen-independent carry-over
Specimen-independent carry-over represents another
type of carry-over which can be caused by diluent
(diluent carry-over) or by reagents (reagent carry-over).
Reagent probe carry-over may for example, be encountered
in analysers where the same reagent probe is used to
dispense reagents from two or more reagent containers,
on a random basis.
The reagent probe carry-over can be determined from the
following sequence using the same specimen and different
methods (identified by letters)"
al, a2, a3, a4, a5, b, a6, c, a7, d, a8, e, a9, alo, all, a12, a13, a14.
182
R. Haeckel Recommendations for definition and determination of carry-over effects
The reagent probe carry-over from reagents ofmethod b
is calculated according to equation (6):
deviates from its correct value which is obtained in the
absence of carry-over effects.
hb a6- d (6)
The error in a6 (a7, as, a0) arising from carry-over due to
reagent x in test b (or c, d, e) can be ignored if the value of
a6 to a0 is less than the mean value ofd + 2s: ofthe values
determined in specimen a.
The sequence must be analysed for each method (a-e in
the example chosen) performed on the particular multitest
analyser and should be repeated at least once unless
the instrument design makes some reagents interactions
impossible. Marked effects have been reported for
carry-over, for example between reagents for the lipase
and triglyceride assays [3]. The specimen used should
have a mid-range concentration of all the analytes to be
measured so that both inhibition and enhancement
effects can be detected [4].
Developments of new analytical systems should avoid
any type of carry-over as far as possible. For this reason
the concept realized by Technicon in the RA 1000 should
be mentioned as an excellent example which should be
followed by other companies and stimulate further
improvements. The patented principle is the application
of an immiscible liquid called Technicon random access
fluid. This is a fluorocarbon and coats the internal and
external surface of the sample and the reagent probe to
prevent sample or reagent contact with the probe walls. A
single probe is used for the aspiration and dispensing of
all samples while another handles all reagents.
This principle has been further developed to the so-called
capsule flow technique applied in the new Chem-1
analyser which seems to introduce a new generation of
exciting analytical systems.
Conclusion
The concept of presenting carry-over effects as a difference
oftwo results, or its corresponding percentage value,
has two advantages:
(1) It is applicable to all conditions which are currently
found in practice.
(2) It directly reports on how much a particular result
References
1. HAECKEL, R., Publication in preparation.
2. HAECKEL, R., and PORTH, J. A., Journal ofClinical Chemistry
and Biochemistry, 10 (1972), 91.
3. HA.CKEL, R., Journal ofClinical Chemistry and Biochemistry, 23
985), 5.
4. BROUOnTON, P. M. G., GOWNLOCK, A. H., McCoRMACK,J.J.
and N.XLL, D. W., Annals ofClinical Biochemistry, 11 (1974),
207.
TECHNICON H* l/H6000 USERS’ MEETING
The 1988 H*I/H6000 Users’ Meeting was held in Oxford, UK. Users were brought up-to-date with the latest modifications for the H*I
these included an IDEE reader, automatic sampler and additional data storage. David Powell fromJ. S. Pathology Services then related two
years’ comparison ofthe two H* ls, the H6000 and two Coulters on that site. The ’Dumping Provocation Test’ was the topic for Neil Porter’s
presentation. He has used the H6000 at the Royal Hallamshire Hospital to monitor the sudden decrease in PCV that can occur in some
patients following gastric surgery. Dr Martin Rowan ofthe Western Infirmary, Glasgow, spoke about the validity ofthe MCV and MCHC
and Keith Morris from Coventry gave results ofa study relating to the use ofthe H* in iron deficiency and spherocytosis. DrJohn Van de
Pette gave the first ofthree presentations from Frimley Park Hospital where an H* Junior is installed. He referred to the use ofthe H* red
cell data to monitor reticulocyte response in megalobalstic anaemia. His colleagues,James Newhouse and Tony Bateman, then gave papers
on the laboratory’s assessment ofthe H* Junior and PCV measurements in iron deficient red cells. The day finished with presentations on
monitoring the progress of AIDS patients and measuring haemoglobin in the presence of gross leucocytosis.
The first paper of the second day was presented by Mike Watts from The Middlesex Hospital who had spent hours doing manual
differentials on leucopenic samples and compared them with the results obtained from the H* 1. Following on from this, DrJohn Richards
from University College Hospital spoke ofthe blast flag response after bone marrow transplantation. Sheila Worthington from George Eliot
Hospital described tracking down the members ofa family in Nuneaton who exhibited beta thalassaemia trait. The final H* User to speak
was Tom Cavanagh from Glasgow who presented a comparison of the three part and five part differentials.
Further detailsfiom Technicon.
183
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