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The validation of biochemical assays by recovery data with special reference to serum iron
CLINICA
THE
CHIMICA
VALIDATION
WITH
SPECIAL
R. G. RYALL
229
ACTA
OF BIOCHEMICAL REFERENCE
BY
RECOVERY
DATA
IRON
AND J. FIELDING
Department of Haematology, (Received
ASSAYS
TO SERUM
February
St. Mavy’s Hospital, Harrow Road, London, W.9 (U.K.)
25, 1971)
SUMMARY
A procedure is described which may be used to validate biochemical analyses based on protein precipitation. It is divided into two stages in which the recovery of added substrate is estimated by isotopic and calorimetric analysis and compared with similar recovery from standards. It is sensitive and simple to perform, information on the source and magnitude of procedural errors.
and provides
INTRODUCTION
There are assumptions on which the validity of biochemical assays depend, which are not always recognised. The validation of an assay procedure using recovery data or other procedures, is governed by two conditions, and compliance with these conditions must be demonstrated. In the following, serum iron assay with which we have been primarily concerned, is used as an example. Condition I: The distribution of iron in the test specimen must at all times during the analysis correspond to the distribution of iron in the standard. This ensures that the result from the test sample can be read directly from the standard.
against
the result obtained
Condition 2: The iron in the standard and the iron in the test must respond equally to the means of measuring them. Microgram for microgram they must, for example, give the same intensity of colour when measured spectrophotometrically. These two conditions must be met if a test procedure is to be considered valid. A further condition of validation from recovery data, already establishedl, is that added iron and native iron behave identically. METHOD
The following reagents are required: (I) All the reagents for the method under investigation. (2) A conveniently 59Fe-labelled FeCl, aqueous solution 20 pug/ml-l.
of approximately
Clin. Chim. Acta, 33 (1971) zzg--234
RYALL, FIELDING
230 (3) Test serum. The procedure is divided into two steps corresponding stated
Testing
to the two conditions
above.
ofCondition
I
In this first step the activity of added 59Fe is used to determine the distribution of iron in the test sample in order to compare it with the distribution of iron in the standards. Increments of 0.01 ml of the 59Fe-labelled FeC1, solution are added to a series of 2.0 ml aliquots of the test serum, starting with o, 0.01, . ..o.os ml. All volumes are made up to 2.05 ml with deionised water. These small increments are sufficient to raise the serum iron level by approximately IO ,ug per increment IOO ml-l without appreciably diluting the serum. Standing the test samples at bench temperature for 30 min permits the iron to bind to transferrin. Identical increments of the same labelled FeCI, solution are added to a second series of 2.0 ml aliquots of standard iron solution. All volumes are made up to 2.05 ml as before. The analysis method is applied to each series and taken as far as precipitation of proteins and the removal of a supernatant aliquot for analysis. Care is taken not to lose any of the liquid phase during this manipulation. The 59Fe activity in the supernatant aliquot is counted and plotted against the 59Fe activity counted in the residue fraction. Fig. I shows the theoretical patterns which could arise from different butions of iron between the supernatant aliquots and their residue fractions.
distri-
63 c
“Fe
in supernotont
ollquot (arbitrary units)
Fig. 1. Theoretical patterns of distribution of “9Fe between supernatant and residue fractions. Slope a: due to coprecipitation of iron with proteins; slope b: volume expansion; slope c: volume contraction; slope d: slope given by standards corresponding to the ratio of volumes of the two fractions.
In a valid method the distribution of the iron between the supernatant and residue fractions corresponds exactly to the distribution observed in the standards. For the aqueous standards the graph has a theoretical slope corresponding to the ratio of the volumes of the two fractions. Deviations from this slope in the test specimens may arise from several causes: the incomplete release of iron from transferrin
VALIDATION
OF BIOCHEMICAL
231
ASSAYS
or the adsorption of iron on to the precipitate phase will all produce relatively more activity
or a volume expansion in the residue fraction
of the fluid than in the
supernatant. Conversely, a contraction in volume would tend to concentrate this fraction. We have observed deviations of the test curve from the standard curve when testing various iron analysis methods. Fig. 2 (slope a) shows the distribution of iron observed using the classical technique of Bothwell and Mallett2 with stored and frozen specimens. An excess of 5gFe activity is clearly demonstrated in the residue ; this has been shown to be due to the adsorption of ferric ions by the precipitate3. Slope b shows an example of the curve given when iron-transferrin dissociation was incomplete due to the omission of additional acid prior to protein precipitation. In these cases the distribution of iron in the test specimen does not agree with the distribution in the standard, and the assay method is in error.
Fig. 2. Testing of condition I under three different circumstances. Slope a (u) due to coprecipitation of iron with protein precipitate; slope b (0) due to incomplete release of iron: slope c (A) shows close agreement between standards and test. Serum iron assay following protocol of Expert Panel on Iron, International Committee for Standardisation in Haematology. Solid line shows slope of standards.
Fig.
2
also shows the results
obtained
using the protocol
for serum iron assay
recommended by the Expert Panel on Iron of the International Committee for Standardization in Haematology*. Here the standard and test curves almost agree though it can be seen that this method gives a result which is slightly too high. This consistent finding is due to a small diminution in volume of the fluid phase as a result of TCA protein precipitation5. Of all methods used in this laboratory, this gave the closest agreement between standard and test curves; the disparity amounting to approximately +3.5% inherent in TCA protein precipitation5, was the mean value for all sera tested. Testing for Condition z If there is agreement between standards and test specimens in respect of condition I, it remains to show that the iron in both series responds equally to colorimetric measurement. Clin. Chim. Acta,
33 (1971) 229-234
RYALL,
232 TABLE:
FIELDING
I
CALCULATION
OF
Total counts
COUNTS
PER
UNIT
IN
ABSORBAh-CE
InCVeaSC
Counts @v absovbance incrrasr
0.275 0.293 0.315 0.346 0.382
0.035 0.053 0.075 0.106
99 IO2
0.142
IOj
0.391
O.IjI
III
Absorbance measured
0
INCREASE
Mean
0.240
3474 5392 9595 11113 14921 16691
I28
108
IO5
Table I shows the absorbance values observed for a series of standards estimated calorimetrically at 535 nm using bathophenanthroline sulfonate as the chromogen. The observed absorbance of the standards increased with increasing iron concentration as shown in column 2. The 5@Fe activity added initially to each tube and assessed by the total number of counts per unit time, also increases and is listed in column I. An increase in absorbance therefore corresponds to an increase in Ve activity, and a mean figure for the number of counts per 0.001 increase in absorbance can be deduced (Column 3). Knowing the s9Fe activity of the test specimens, it is therefore possible to calculate an expected increase in absorbance for each of these which is then compared with the observed increase. Fig. 3 shows the theoretical possibilities when the calculated increase in absorbance is plotted against the observed increase.
Fig. 3. Theoretical patterns of response of iron to calorimetric measurement. >lope a: due to colour inhibition; slope b: correct response : slope c : due to colour enhancement; slope d : due to inclusion of contaminant in assay results.
A valid test will give a line of unit slope. Colour inhibition by a competing complexing agent results in a smaller increase in observed absorbance when compared with that calculated. The converse of this, a colour promoter, has not been observed experimentally and can be considered of only theoretical interest in iron methods. Contamination by other sources of iron may be detected if a third series, similar to the test series, is set up initially and to which is added the suspected contaminant. Clin. Chim.
Acta,
33 (1971)
229-234
VALIDATION
OF BIOCHEMICAL
Comparison
of the observed
gives a line cutting enters
233
ASSAYS
absorbance
values
with
the
one of the axes, if the contaminating
un-contaminated
series
source (e.g. haemoglobin)
into the assay procedure.
Fig. 4. a, Testing of Condition I for titrated plasma shows close agreement between standards and test. b, Testing of Condition 2 shows discrepancy between standards and test (a) due to colour inhibition in titrated plasma (A) ; (c) d ue t o inclusion of haemoglobin iron in the assay result (a) : (b) uncontaminated sample (n) shows agreement between standards and test.
Fig. 4 shows the results obtained in the analysis of titrated plasma by the modified method of Bothwell and MalletP. Step I (Fig. 4a) of the validation showed that the distribution of iron in the test agreed with that in the standards. Colour development was partially inhibited, however, as shown in the corresponding graph for step 2 (Fig. 4b, slope a). Agreement between standards and test specimens was not found and this method is therefore not valid for the analysis of titrated plasmas. Fig. 4 also shows the results of the analysis of samples with added haemoglobin by the same method. From step I of the validation it was found that the presence of haemoglobin did not affect distribution of iron in the test samples and that this agreed closely with the standards. In step 2 however, the plot of observed increases in absorbance against those calculated, showed that some haemoglobin iron was being included in the assay (slope c, Fig. 4b). The analysis of an uncontaminated series is included for comparison (slope b, Fig. 4b). DISCUSSION
By far the most common means of validating methods for serum iron has been by the analysis of recovery data. Between 2 and 400 pg of iron IOO ml-l of serum have been added to test samples276 and the correct estimation of this iron has led to a claim of validity for the method used. A wide variety of ferrous and ferric salts and chelates have been used, and there has been as wide a range of recovery techniques as the methods they attempt to validate. No two authors follow the same procedure. Sometimes incomplete recovery of added iron has led to the inclusion of a correction factor in the calculation instead of an amendment to the analysis protocol’. Comparison with other techniques8 and the dilution of serum samples to show known fractions of a previously determined totals have also been used for validation. The procedure described here for the validation of serum methods is one which Clin.
Chim.
Acta,
33 (1971)
zzg--234
RYALL,
234
FIELDING
enables the validity of a given analysis protocol to be assessed rapidly and with great sensitivity. Agreement between standards and test specimens in both stages of the procedure must be demonstrated for validation. Failure to agree means error in the analysis. The procedure demonstrates the points at which errors arise and indicates their magnitude. One aspect of an analysis method which is not tested in this way is the validation of the blanks used in the assay. This cannot be determined from recovery data and there is to date no method of validating blanks in a given protocol. In clinical biochemical methods there seems to be an inclination to accept techniques which give the highest results. The validation procedure described here shows that a technique may give results which are too high and there is no justification for accepting methods giving the highest values as the most accurate. REFERENCES I 2 3 4 5 6 7 8 9
D’A. KOK AND F. WILD, .I. Clin. P&hot., 13 (1960) 241. T. BOTHWELL AND B. MALLETT, Biochem. ,J., 59 (1955) 599. R. RYALL AND J. FIELDING, Clin. Chim. Acta, 28 (1970) 193. International Committee for Standardisation in Haematology, Brit. J. Haematol., 20 (1971) 4 jr. J. FIELDING AND R. RYALL, Clin. Chim. Acta, 33 (1971) 237. A. SCH~DE, J. OYAMA, R. REINHART AND J. MILLER, Proc. Sot. Exp. Biol. Med., 87 (1954) 443. H. BURCH, 0. LOWRY, 0. BESSEY API’D B. BERSON, ./. Biol. Chem., 174 (1948) 791. W. N. M. RAMSAY, Clin. Chim. Acta, 2 (1957) 214. W. T. CARAWAY, Clin. Chem., 9 (1963) 188.
Cli?%.ChiTI?..Acta,
33 (1971) 229-234
THE
CHIMICA
VALIDATION
WITH
SPECIAL
R. G. RYALL
229
ACTA
OF BIOCHEMICAL REFERENCE
BY
RECOVERY
DATA
IRON
AND J. FIELDING
Department of Haematology, (Received
ASSAYS
TO SERUM
February
St. Mavy’s Hospital, Harrow Road, London, W.9 (U.K.)
25, 1971)
SUMMARY
A procedure is described which may be used to validate biochemical analyses based on protein precipitation. It is divided into two stages in which the recovery of added substrate is estimated by isotopic and calorimetric analysis and compared with similar recovery from standards. It is sensitive and simple to perform, information on the source and magnitude of procedural errors.
and provides
INTRODUCTION
There are assumptions on which the validity of biochemical assays depend, which are not always recognised. The validation of an assay procedure using recovery data or other procedures, is governed by two conditions, and compliance with these conditions must be demonstrated. In the following, serum iron assay with which we have been primarily concerned, is used as an example. Condition I: The distribution of iron in the test specimen must at all times during the analysis correspond to the distribution of iron in the standard. This ensures that the result from the test sample can be read directly from the standard.
against
the result obtained
Condition 2: The iron in the standard and the iron in the test must respond equally to the means of measuring them. Microgram for microgram they must, for example, give the same intensity of colour when measured spectrophotometrically. These two conditions must be met if a test procedure is to be considered valid. A further condition of validation from recovery data, already establishedl, is that added iron and native iron behave identically. METHOD
The following reagents are required: (I) All the reagents for the method under investigation. (2) A conveniently 59Fe-labelled FeCl, aqueous solution 20 pug/ml-l.
of approximately
Clin. Chim. Acta, 33 (1971) zzg--234
RYALL, FIELDING
230 (3) Test serum. The procedure is divided into two steps corresponding stated
Testing
to the two conditions
above.
ofCondition
I
In this first step the activity of added 59Fe is used to determine the distribution of iron in the test sample in order to compare it with the distribution of iron in the standards. Increments of 0.01 ml of the 59Fe-labelled FeC1, solution are added to a series of 2.0 ml aliquots of the test serum, starting with o, 0.01, . ..o.os ml. All volumes are made up to 2.05 ml with deionised water. These small increments are sufficient to raise the serum iron level by approximately IO ,ug per increment IOO ml-l without appreciably diluting the serum. Standing the test samples at bench temperature for 30 min permits the iron to bind to transferrin. Identical increments of the same labelled FeCI, solution are added to a second series of 2.0 ml aliquots of standard iron solution. All volumes are made up to 2.05 ml as before. The analysis method is applied to each series and taken as far as precipitation of proteins and the removal of a supernatant aliquot for analysis. Care is taken not to lose any of the liquid phase during this manipulation. The 59Fe activity in the supernatant aliquot is counted and plotted against the 59Fe activity counted in the residue fraction. Fig. I shows the theoretical patterns which could arise from different butions of iron between the supernatant aliquots and their residue fractions.
distri-
63 c
“Fe
in supernotont
ollquot (arbitrary units)
Fig. 1. Theoretical patterns of distribution of “9Fe between supernatant and residue fractions. Slope a: due to coprecipitation of iron with proteins; slope b: volume expansion; slope c: volume contraction; slope d: slope given by standards corresponding to the ratio of volumes of the two fractions.
In a valid method the distribution of the iron between the supernatant and residue fractions corresponds exactly to the distribution observed in the standards. For the aqueous standards the graph has a theoretical slope corresponding to the ratio of the volumes of the two fractions. Deviations from this slope in the test specimens may arise from several causes: the incomplete release of iron from transferrin
VALIDATION
OF BIOCHEMICAL
231
ASSAYS
or the adsorption of iron on to the precipitate phase will all produce relatively more activity
or a volume expansion in the residue fraction
of the fluid than in the
supernatant. Conversely, a contraction in volume would tend to concentrate this fraction. We have observed deviations of the test curve from the standard curve when testing various iron analysis methods. Fig. 2 (slope a) shows the distribution of iron observed using the classical technique of Bothwell and Mallett2 with stored and frozen specimens. An excess of 5gFe activity is clearly demonstrated in the residue ; this has been shown to be due to the adsorption of ferric ions by the precipitate3. Slope b shows an example of the curve given when iron-transferrin dissociation was incomplete due to the omission of additional acid prior to protein precipitation. In these cases the distribution of iron in the test specimen does not agree with the distribution in the standard, and the assay method is in error.
Fig. 2. Testing of condition I under three different circumstances. Slope a (u) due to coprecipitation of iron with protein precipitate; slope b (0) due to incomplete release of iron: slope c (A) shows close agreement between standards and test. Serum iron assay following protocol of Expert Panel on Iron, International Committee for Standardisation in Haematology. Solid line shows slope of standards.
Fig.
2
also shows the results
obtained
using the protocol
for serum iron assay
recommended by the Expert Panel on Iron of the International Committee for Standardization in Haematology*. Here the standard and test curves almost agree though it can be seen that this method gives a result which is slightly too high. This consistent finding is due to a small diminution in volume of the fluid phase as a result of TCA protein precipitation5. Of all methods used in this laboratory, this gave the closest agreement between standard and test curves; the disparity amounting to approximately +3.5% inherent in TCA protein precipitation5, was the mean value for all sera tested. Testing for Condition z If there is agreement between standards and test specimens in respect of condition I, it remains to show that the iron in both series responds equally to colorimetric measurement. Clin. Chim. Acta,
33 (1971) 229-234
RYALL,
232 TABLE:
FIELDING
I
CALCULATION
OF
Total counts
COUNTS
PER
UNIT
IN
ABSORBAh-CE
InCVeaSC
Counts @v absovbance incrrasr
0.275 0.293 0.315 0.346 0.382
0.035 0.053 0.075 0.106
99 IO2
0.142
IOj
0.391
O.IjI
III
Absorbance measured
0
INCREASE
Mean
0.240
3474 5392 9595 11113 14921 16691
I28
108
IO5
Table I shows the absorbance values observed for a series of standards estimated calorimetrically at 535 nm using bathophenanthroline sulfonate as the chromogen. The observed absorbance of the standards increased with increasing iron concentration as shown in column 2. The 5@Fe activity added initially to each tube and assessed by the total number of counts per unit time, also increases and is listed in column I. An increase in absorbance therefore corresponds to an increase in Ve activity, and a mean figure for the number of counts per 0.001 increase in absorbance can be deduced (Column 3). Knowing the s9Fe activity of the test specimens, it is therefore possible to calculate an expected increase in absorbance for each of these which is then compared with the observed increase. Fig. 3 shows the theoretical possibilities when the calculated increase in absorbance is plotted against the observed increase.
Fig. 3. Theoretical patterns of response of iron to calorimetric measurement. >lope a: due to colour inhibition; slope b: correct response : slope c : due to colour enhancement; slope d : due to inclusion of contaminant in assay results.
A valid test will give a line of unit slope. Colour inhibition by a competing complexing agent results in a smaller increase in observed absorbance when compared with that calculated. The converse of this, a colour promoter, has not been observed experimentally and can be considered of only theoretical interest in iron methods. Contamination by other sources of iron may be detected if a third series, similar to the test series, is set up initially and to which is added the suspected contaminant. Clin. Chim.
Acta,
33 (1971)
229-234
VALIDATION
OF BIOCHEMICAL
Comparison
of the observed
gives a line cutting enters
233
ASSAYS
absorbance
values
with
the
one of the axes, if the contaminating
un-contaminated
series
source (e.g. haemoglobin)
into the assay procedure.
Fig. 4. a, Testing of Condition I for titrated plasma shows close agreement between standards and test. b, Testing of Condition 2 shows discrepancy between standards and test (a) due to colour inhibition in titrated plasma (A) ; (c) d ue t o inclusion of haemoglobin iron in the assay result (a) : (b) uncontaminated sample (n) shows agreement between standards and test.
Fig. 4 shows the results obtained in the analysis of titrated plasma by the modified method of Bothwell and MalletP. Step I (Fig. 4a) of the validation showed that the distribution of iron in the test agreed with that in the standards. Colour development was partially inhibited, however, as shown in the corresponding graph for step 2 (Fig. 4b, slope a). Agreement between standards and test specimens was not found and this method is therefore not valid for the analysis of titrated plasmas. Fig. 4 also shows the results of the analysis of samples with added haemoglobin by the same method. From step I of the validation it was found that the presence of haemoglobin did not affect distribution of iron in the test samples and that this agreed closely with the standards. In step 2 however, the plot of observed increases in absorbance against those calculated, showed that some haemoglobin iron was being included in the assay (slope c, Fig. 4b). The analysis of an uncontaminated series is included for comparison (slope b, Fig. 4b). DISCUSSION
By far the most common means of validating methods for serum iron has been by the analysis of recovery data. Between 2 and 400 pg of iron IOO ml-l of serum have been added to test samples276 and the correct estimation of this iron has led to a claim of validity for the method used. A wide variety of ferrous and ferric salts and chelates have been used, and there has been as wide a range of recovery techniques as the methods they attempt to validate. No two authors follow the same procedure. Sometimes incomplete recovery of added iron has led to the inclusion of a correction factor in the calculation instead of an amendment to the analysis protocol’. Comparison with other techniques8 and the dilution of serum samples to show known fractions of a previously determined totals have also been used for validation. The procedure described here for the validation of serum methods is one which Clin.
Chim.
Acta,
33 (1971)
zzg--234
RYALL,
234
FIELDING
enables the validity of a given analysis protocol to be assessed rapidly and with great sensitivity. Agreement between standards and test specimens in both stages of the procedure must be demonstrated for validation. Failure to agree means error in the analysis. The procedure demonstrates the points at which errors arise and indicates their magnitude. One aspect of an analysis method which is not tested in this way is the validation of the blanks used in the assay. This cannot be determined from recovery data and there is to date no method of validating blanks in a given protocol. In clinical biochemical methods there seems to be an inclination to accept techniques which give the highest results. The validation procedure described here shows that a technique may give results which are too high and there is no justification for accepting methods giving the highest values as the most accurate. REFERENCES I 2 3 4 5 6 7 8 9
D’A. KOK AND F. WILD, .I. Clin. P&hot., 13 (1960) 241. T. BOTHWELL AND B. MALLETT, Biochem. ,J., 59 (1955) 599. R. RYALL AND J. FIELDING, Clin. Chim. Acta, 28 (1970) 193. International Committee for Standardisation in Haematology, Brit. J. Haematol., 20 (1971) 4 jr. J. FIELDING AND R. RYALL, Clin. Chim. Acta, 33 (1971) 237. A. SCH~DE, J. OYAMA, R. REINHART AND J. MILLER, Proc. Sot. Exp. Biol. Med., 87 (1954) 443. H. BURCH, 0. LOWRY, 0. BESSEY API’D B. BERSON, ./. Biol. Chem., 174 (1948) 791. W. N. M. RAMSAY, Clin. Chim. Acta, 2 (1957) 214. W. T. CARAWAY, Clin. Chem., 9 (1963) 188.
Cli?%.ChiTI?..Acta,
33 (1971) 229-234