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Empirical relations as interference correctives in multichannel analyzers
Clinica Chimica Acta, 60 (197 5) 7-l 3 @ Elsevier Scientific Publishing Company,
Amsterdam
-
Printed
in The Netherlands
CCA 6867
EMPIRICAL RELATIONS AS INTERFERENCE MULTICHANNEL ANALYZERS
HENNING
IN
BOKELUND
Department of Clinical Odense (Denmark) (Received
CORRECTIVES
October
Chemistry,
Odense
University
Hospital,
DK-5000
1, 1974)
Summary Interference from turbidity and bilirubin on 20 serum constituents have been examined on the AutoChemist multichannel analytical system. Empirical relations are presented which correct for these effects on tests such as acid phosphatase, alkaline phosphatase, bilirubin, chloride, cholesterol, creatinine, iron, lactate dehydrogenase, phosphate and uric acid. The interference correctives are routinely applied to patient specimens by aid of the computer attached to the system. Introduction Among the specimens presented for analysis in the clinical chemistry laboratory some may be considered especially error prone due to the presence of haemolysis, lipaemia or bilirubinemia. Some laboratories recommend the rejection of specimens with visible haemolysis and abstain from the determination of about 14 consituents in plasma under such conditions [l] . Lipaemic sera cause problems in the calorimetric determination of several serum constituents in that the absorbance may be falsely elevated in the presence of turbidity. This interference is not always adequately compensated for by blank procedures [2] ; moreover many analytical methods do not include individual serum blanks. Serum obtained from non-fasting subjects presents a special problem in this respect, in that the turbidity caused by chylomicrons will persist in the serum several hours after the meal [3] and will render such determinations as e.g. thymol turbidity highly unreliable [4] . The effects of icteric sera on calorimetric procedures may be of two kinds: (i) The colour of the end product of the chemical reaction used in the procedure coincides with the colour of the bilirubin in the specimen (spectral interference).
8
(ii) Bilirubin or its protein complexes interact in the chemical reaction used for the determination of the constituent in question or it forms coloured complexes with the reagents (chemical interference). The influence of high serum bilirubin values has been reported on cholesterol in the Liebermann-Burchard reaction [5], on the assay of acid phosphatase using oc-naphthyl phosphate as substrate according to Babson and Phillips [6] , on the determination of uric acid by uricase methodology [7], and on serum iron by the nitroso-R procedure [8]. In this study empirical relations are presented which correct for the influence of turbidity and bilirubinemia on serum constituents such as chloride, inorganic phosphate, iron, creatinine, acid and alkaline phosphatases, lactate dehydrogenase, uric acid and cholesterol. The relations are of the linear form: AY = a + bX, where AY is the term to be deducted from or added to the result of the measurement of the constituent Yin order to yield a value corrected for the influence of X, which in this case can be either turbidity or bilirubin concentration. The coefficients a and b are found from regression analysis. The basic measurements are performed on the AutoChemist@ multichannel analytical system (AutoChem Instrument AB, Lidingo, Sweden), which in our case is equipped for the measurement of 20 serum constituents (sodium, potassium, calcium, chloride, phosphate, urea, creatinine, total protein, albumin, iron, uric acid, total lipids, cholesterol, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline and acid phosphatases, thymol turbidity and total bilirubin), employing individual blanks for 5 of these. In addition to the methods used on the AutoChemist, other methods e.g. involving dialysis have been used to determine A Y (see below). The relations are stored in the AutoChemist PDP 12 computer (Digital Equipment Corporation, Maynard, Mass., U.S.A.) and are thus used as correctives on measurements of patient sera performed on the system. Methods and Materials The chemistry methods used on our AutoChemist are referred to elsewhere [ 91. They include the methods referred to above [ 4-81 and the method of Babson and Phillips for lactate dehydrogenase [lo] . The blank channel used for serum iron in the methodology of Ness and Dickerson [8] was used as an index of turbidity. The procedure uses sodium acetate as a diluent on the serum and photometry at 720 nm. Turbidity is thus for this purpose expressed in arbitrary units (coherent with units for serum iron, i.e. pmol/l). Most of the patient sera have turbidity values of l----3 units; however, elevated values of up to about 35 units are found on sera with pronounced lipaemia. In order to obtain a relationship between the turbidity, T, measured on the AutoChemist, as explained above, and its interference with the results of other channels (such as chloride, phosphate (P, inorganic), iron, bilirubin and creatinine) many patient sera with turbidities above 5 units were, in addition to the analyses on the AutoChemist, analyzed by other techniques not sensitive to turbidity. The differences, AY, found as the AutoChemist result minus the result from the other procedures, was then related to T by linear regression analyses (AY = a + 0 T).
9
Chloride was titrated on the Radiometer chloride titrator “CMT lo”, with a silver electrode and potentiometric endpoint [ 111. Phosphate was determined on the Technicon Autoanalyser using a dialysis/molybdenum blue method [12], iron was assayed using dialysis and the Autoanalyzer bathophenanthroline procedure [ 131, whereas creatinine was analyzed after dialysis by the Jaffe-Autoanalyzer methodology [ 141. For bilirubin another approach was taken: From our participation in an external quality control scheme employing weekly samples with grossly varying turbidity, AY was established as the difference(s) between the found value(s) and the mean(s) of the results from the 12 participating laboratories. A crucial point in the above mentioned comparisons between the results from the AutoChemist and from other methods in order to establish AY values is the absence of bias between methods. As a part of our internal quality control system this condition is verified routinely by comparing the results of daily measurements of control sera by both methods. On a monthly basis, t tests are performed on the means to test the hypotheses of a difference between methods of zero. The empirical relationships in the case of icteric sera has the same format, AY = a + b B, where B denotes the concentration of bilirubin in pmol/l. They are based on three sets of measurements of bilirubin preparations in albumin (Dade), each set consisting of a series of dilutions using albumin solutions as diluent. The range of bilirubin concentrations was 36-315 pmol/l. All measurements were performed on the AutoChemist, in that the results on all 20 main channels and the 5 blank channels were recorded, forming the basis for the evaluation of AY for each constituent. As above, linear regressions of “AY” on “B” yield the coefficients “a” and “b”, to be used as future correctives for bilirubinemia via the computer. Results Turbidity corrections Table I shows the coefficients a and b of the regression lines, along with the correlation coefficients, r, and the standard deviations of estimates of AY at the level of turbidity of 10 units, as calculated from the residual variance TABLE
I
CORRECTIONS
FOR
LIPEMIA
b
S(A
r
cl
YIO)*
% of AYIO
Chloride
(mmol/l)
Phosphate Iron
0.45
(mmol/l)
0.020
Creatinine
* S(A
0.402
8.0
0.981
0.0070
4.3 3.2
0.53
0.992
0.643
0.06
0.972
0.360
7.3
-5.10
3.25
0.990
1.39
2.6
1.95
(fimol/l)
0.987
-0.50
(firnOl/l)
Bilirubin
0.51 --a.035
(~mol/l)
Y IO) is the errw
of A Y at T = 10 units,
calculated
line. **
S(A
Ylo)
expressed
in percent
of AY
at T = 10 units.
from
the residual
error
around
**
the regression
TABLE
II
QUALITY
CON’I‘KOL
COMPAKISONS
Period M.D.’
Chloride
(mmol/l)
Phosphate Iron
(mnol/I)
(pmol/l)
Crrdtininr
1
Period li*
of the
M.D.
f
3
M.D.
1
1.52
n.s.
0.3
0.43
n.s.
I.7
1.69
11,s.
n.s.
0.02
1.42
11,s.
~0.04
1.23
11.s.
-0.4
I .03
n.s.
-0.2
0.54
11,s.
0.7
1.29
n.s.
0.82
11,s.
--2.8
1.38
11.s.
1.0
0.58
n.s.
I.2
?i measurements, not
Period
1.61
denotesdifference
n.s.,
2
-0.9
between
the rman
t = IS, -x_? l/j(( nl - 1)s: + (n2 ~ I)& tively.
METHODS
-0.02
(pmol/l)
(i M.D. * +
BETWEEN
significant
subscripts
“1”
at a 1, = 0.05
and
of
the
AutoChemist
and
the
01~+ ~I~)/(“~~I~)(I~~ + “2 “2”
refer
significance
to
the
AutoChemist
other 2).
method. where
and
other
i.e.
S’
-YI
s-2.
1s the variance
method.
respec-
level
around the regression line [ 151 . In the last column these standard deviations are related to AY, 0 (the AY at T = 10 units) and expressed as percentages. The errors of the corrections amount to 2----8% of the corrections, which is considered highly acceptable, e.g. an uncorrected value of 70 pmol/l of creatinine at a turbidity of 10 units will be corrected into 124 pmol/l with an error of i- 1 pmol/l (apart from the error involved in the measurement of the uncorrected 70 pmol/l). The significance of the intercept a is verified by the calculation of its error from the residual variance around the regression line. In all cases the 95% confidence interval around a was found to include the value zero; a is thus not significantly different from zero, as also predicted by theory (AY = 0 for T = 0). In the case of bilirubin and creatinine the correction is in the direction which increases the results, as the turbidity interference tends to elevate the blank values. Table II shows the mean differences between methods found from the internal quality control prograrn. Also given are the t values for three onemonth periods. The outcome of the t tests on the difference between AutoChemist and other methodologies was in all cases not statistically significant at the 95% confidence level. This supports the validity of the empirical relations in which AY is the difference between results found on the AutoChemist and from other methods, in that no significant bias exists between the basic measurement methods. The effect of the introduction of the correctives in routine use can be appraised from Fig. 1, which shows results from an external quality control programme. The graph is a cusum presentation of phosphate analysis. The ordinate represents the cusum of the percentage difference(s) between the value(s) of our laboratory and the reference value(s) for each weekly sample. The actual turbidity values in each sample are indicated on the graph; it is seen how high turbidity values coincide with jumps on the cusum curve. Obviously, the artifact of turbidity is removed as from the introduction of the correction.
11
of dtfferences
Curum
PHOSPHATE
Fig.
1.
Cusum
between sample
presentation
result(s) is
found
noted
on
of and
the
week
20
IO quality
reference
curve.
The
control
results
value(s) cusum
on phosphate
are summed curve
no
levels
on off
analyses.
the
Y-axis.
as from
the
The The
percentage turbidity
difference(s) in each
introduction
of
the
weekly
turbidity
correction.
Bilirubin corrections Table III shows in a similar manner to Table I the coefficients a, b, r for the case of bilirubinemia. From the residual variance around the regression line error estimates of AY are calculated at a bilirubin level of 80 pmol/l (approximately four times the upper limit of normals). These standard deviations and their percentages of AYs 0 (the AY value at B = 80 pmol/l) are given in the last two columns of Table III. Again the errors of AY are small (l--10%) and again a verification of the significance of a shows a to be not significantly different from zero, except in the case of alkaline phosphatase, where a significant intercept of 0.7 U/l was found. The random error given in Table III for alkaline phosphatase is extremely small (note r = 1.000); but in addition a systematic error component of 0.7 U/l is present. In view of the reference range for alkaline phosphatase of 29-88 U/l this bias is negligible. TABLE
III
CORRECTIONS
FOR
BILIRUBINEMIA
b
r
a
.s(A
Y&*
$2 of AY80
Chloride Iron
(mmolil)
(pmol/l)
Cholesterol Acid
(mmol/l)
phosphatase
Alkaline Uric
acid,
blank
Creatinine. LDH.
(U/l)
phosphatase
blank
blank
* * S(A
(mmol/I) (pmol/I)
0.988
0.167
0.41
0.999
0.160
0.0058
- 0.08
0.995
0.0389
10.1
0.0103
0.1
0.992
0.0576
3.5
0.036
0.7
1.000
0.0044
0.1
0.00141
0.009
0.998
0.0021~
1.8
0.9998
0.266
0.62
0.999
0.672
1.4
-1.05 1.30
0.58
is the
around Y~o).
0.009
0.107
0.509
(U/I)
* .5’(A Ygg) error
(U/l)
0.041
error
of
the regression
expressed
A Y at a bilimbin
concentration
of 80 pmol/l.
calculated
5.1 1.8
from
line.
as a percentage
of h Y at a bilirubin
concentration
of 80
**
umolil.
the residual
12
In the cases of uric acid, creatinine, and lactate dehydrogenase (LDH) the influence of bilirubin is compensated only in as much as the blank and main channels are equally elevated. The importance of using identical sensitivity settings for the two channels (factor converting signal to medical units) is stressed in this connection. Calibration sera often have very low blank values, the practise of compensating for the low blank signals by increasing the amplifier gain and attenuating the sensitivity factor is not recommendable in view of above interference considerations (optical interference). Discussion It is a unique asset of a multichannel system that all specimens treated can be measured for interfering substances such as e.g. bilirubin or turbidity. The availability of an on-line computer facilitates corrections for interferences. The integration of an on-line computer system in a conventional laboratory is naturally just as adequate for correction purposes provided the basic parameters are measured. The measurement of turbidity at 720 nm is chosen somewhat arbitrarily; however, it leads to an adequate empirical corrective on the tests shown in Table I. Turbidity in serum is caused by particles of varying diameter and concentration as discussed on a theoretical basis by Gautschi and Richterich [ 161, who also describe model experiments using latex suspensions to simulate turbidity. The use of computer corrections for lipemia, and bilirubinemia on the AutoChemist system was introduced by Jungner [17], however, the turbidity corrections were established differently. The influence of bilirubin on cholesterol in the direct LiebermannBurchard procedure was studied by van Boetzelaer and Zondag [18], who found that 1 mg/lOO ml of bilirubin produced the same absorbance as 5 mg/lOO ml of cholesterol measured at 615 nm on the Beckman DU spectrophotometer (equivalent to: b = 0.0076, mmol cholesterol/pmol bilirubin). The correction found in this paper for cholesterol measurements at 595 nm is somewhat smaller (b = 0.0058 * 0.0003). The application of a similar correction for haemolysis based on the haemoglobin concentration is not considered here. Such corrections are probably not recommendable [ 11, as the elevation of the measurement values for certain constituents (K, LDH, etc.) caused by haemolysis may not be an artifact of laboratory procedures but may well be biological. The errors of AY given in Tables I and III are based on the assumption that X in the relation AY = a + bX is found without error. Actually, X has an error, in that T or B are measurements. Error estimates obtained under such conditions tend to overestimate the true error of AY; however, predictions of A Y based on measured X values are valid, in that the random fluctuations of X will be equally present in the data used in the analysis and for prediction [ 191. The general problem discussed in this paper is focused on the individual patient specimen and will not be covered by the use of quality control sera for precision control.
13
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 I.6 17 18 19
W.G. Brydon and L.B. Roberts, Clin. Chim. Acta, 41 (1972) 435 W.T. Caraway, Am. J. Clin. Pathol., 37 (1962) 445 B.E. Statland, P. Wink& and H. Bokelund, CIin. Chem.. 19 (1973) 1380 J.G. Reinhold. Clin. Chem., 8 (1962) 475 T.C. Huang. C.P. Chen, V. Wefler and A. Rafterv. Anal. Chem.. 33 (1961) 1405 A.L. Babson and G.E. Phillips, Clin. Chim. Acta, 13 (1966) 264 S. Morgenstern, R.V. Flor, J.H. Kaufman and B. Klein, Clin. Chem.. 12 (1966) 748 A.T. Ness and H.C. Dickerson, C&n. Chim. Aeta, 12 (1965) 579 B.E. Statland, P. Winkel and H. Bokelund, Clin. Chem., 19 (197-3) 1374 A.L. Babson and G.E. Phillips, Clin. Chim. Acta, 12 (1965) 210 Instrument Manual, Radiometer CMT 10 Chloride Titrator, Radiometer. Copenhagen. Denmark H.Y. Yee, Clin. Chem., 14 (1968) 898 T.J. Giovannieiio, G. Dibenedetto, D.W. Palmer and T. Peters. Jr, J. Lab. Clin. Med.. 71 (1968) 874 Technicon’s Method File: N-11 b Documenta Geigy, Scientific Tables, J.R. Geigy S.A., Basle, 1970, p. 174 M. Gautschi and R. Richterich, 2. Klin. Chem. Klin. Biochem., 11 (1973) 139 I. Jungner, Methods in Clinical Chemistry, Vol. 1 (1970). 7th Int. Congr. Clin. C&m., Geneva, 1969 G.L. van Boetzelaer and H.A. Zondag, Clin. Chim. Acta, 5 (1960) 943 P. Armitage, Statistical Methods in Medical Research, Blackwell Scientific Publications, Oxford, 1971, Section 9.2
Amsterdam
-
Printed
in The Netherlands
CCA 6867
EMPIRICAL RELATIONS AS INTERFERENCE MULTICHANNEL ANALYZERS
HENNING
IN
BOKELUND
Department of Clinical Odense (Denmark) (Received
CORRECTIVES
October
Chemistry,
Odense
University
Hospital,
DK-5000
1, 1974)
Summary Interference from turbidity and bilirubin on 20 serum constituents have been examined on the AutoChemist multichannel analytical system. Empirical relations are presented which correct for these effects on tests such as acid phosphatase, alkaline phosphatase, bilirubin, chloride, cholesterol, creatinine, iron, lactate dehydrogenase, phosphate and uric acid. The interference correctives are routinely applied to patient specimens by aid of the computer attached to the system. Introduction Among the specimens presented for analysis in the clinical chemistry laboratory some may be considered especially error prone due to the presence of haemolysis, lipaemia or bilirubinemia. Some laboratories recommend the rejection of specimens with visible haemolysis and abstain from the determination of about 14 consituents in plasma under such conditions [l] . Lipaemic sera cause problems in the calorimetric determination of several serum constituents in that the absorbance may be falsely elevated in the presence of turbidity. This interference is not always adequately compensated for by blank procedures [2] ; moreover many analytical methods do not include individual serum blanks. Serum obtained from non-fasting subjects presents a special problem in this respect, in that the turbidity caused by chylomicrons will persist in the serum several hours after the meal [3] and will render such determinations as e.g. thymol turbidity highly unreliable [4] . The effects of icteric sera on calorimetric procedures may be of two kinds: (i) The colour of the end product of the chemical reaction used in the procedure coincides with the colour of the bilirubin in the specimen (spectral interference).
8
(ii) Bilirubin or its protein complexes interact in the chemical reaction used for the determination of the constituent in question or it forms coloured complexes with the reagents (chemical interference). The influence of high serum bilirubin values has been reported on cholesterol in the Liebermann-Burchard reaction [5], on the assay of acid phosphatase using oc-naphthyl phosphate as substrate according to Babson and Phillips [6] , on the determination of uric acid by uricase methodology [7], and on serum iron by the nitroso-R procedure [8]. In this study empirical relations are presented which correct for the influence of turbidity and bilirubinemia on serum constituents such as chloride, inorganic phosphate, iron, creatinine, acid and alkaline phosphatases, lactate dehydrogenase, uric acid and cholesterol. The relations are of the linear form: AY = a + bX, where AY is the term to be deducted from or added to the result of the measurement of the constituent Yin order to yield a value corrected for the influence of X, which in this case can be either turbidity or bilirubin concentration. The coefficients a and b are found from regression analysis. The basic measurements are performed on the AutoChemist@ multichannel analytical system (AutoChem Instrument AB, Lidingo, Sweden), which in our case is equipped for the measurement of 20 serum constituents (sodium, potassium, calcium, chloride, phosphate, urea, creatinine, total protein, albumin, iron, uric acid, total lipids, cholesterol, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline and acid phosphatases, thymol turbidity and total bilirubin), employing individual blanks for 5 of these. In addition to the methods used on the AutoChemist, other methods e.g. involving dialysis have been used to determine A Y (see below). The relations are stored in the AutoChemist PDP 12 computer (Digital Equipment Corporation, Maynard, Mass., U.S.A.) and are thus used as correctives on measurements of patient sera performed on the system. Methods and Materials The chemistry methods used on our AutoChemist are referred to elsewhere [ 91. They include the methods referred to above [ 4-81 and the method of Babson and Phillips for lactate dehydrogenase [lo] . The blank channel used for serum iron in the methodology of Ness and Dickerson [8] was used as an index of turbidity. The procedure uses sodium acetate as a diluent on the serum and photometry at 720 nm. Turbidity is thus for this purpose expressed in arbitrary units (coherent with units for serum iron, i.e. pmol/l). Most of the patient sera have turbidity values of l----3 units; however, elevated values of up to about 35 units are found on sera with pronounced lipaemia. In order to obtain a relationship between the turbidity, T, measured on the AutoChemist, as explained above, and its interference with the results of other channels (such as chloride, phosphate (P, inorganic), iron, bilirubin and creatinine) many patient sera with turbidities above 5 units were, in addition to the analyses on the AutoChemist, analyzed by other techniques not sensitive to turbidity. The differences, AY, found as the AutoChemist result minus the result from the other procedures, was then related to T by linear regression analyses (AY = a + 0 T).
9
Chloride was titrated on the Radiometer chloride titrator “CMT lo”, with a silver electrode and potentiometric endpoint [ 111. Phosphate was determined on the Technicon Autoanalyser using a dialysis/molybdenum blue method [12], iron was assayed using dialysis and the Autoanalyzer bathophenanthroline procedure [ 131, whereas creatinine was analyzed after dialysis by the Jaffe-Autoanalyzer methodology [ 141. For bilirubin another approach was taken: From our participation in an external quality control scheme employing weekly samples with grossly varying turbidity, AY was established as the difference(s) between the found value(s) and the mean(s) of the results from the 12 participating laboratories. A crucial point in the above mentioned comparisons between the results from the AutoChemist and from other methods in order to establish AY values is the absence of bias between methods. As a part of our internal quality control system this condition is verified routinely by comparing the results of daily measurements of control sera by both methods. On a monthly basis, t tests are performed on the means to test the hypotheses of a difference between methods of zero. The empirical relationships in the case of icteric sera has the same format, AY = a + b B, where B denotes the concentration of bilirubin in pmol/l. They are based on three sets of measurements of bilirubin preparations in albumin (Dade), each set consisting of a series of dilutions using albumin solutions as diluent. The range of bilirubin concentrations was 36-315 pmol/l. All measurements were performed on the AutoChemist, in that the results on all 20 main channels and the 5 blank channels were recorded, forming the basis for the evaluation of AY for each constituent. As above, linear regressions of “AY” on “B” yield the coefficients “a” and “b”, to be used as future correctives for bilirubinemia via the computer. Results Turbidity corrections Table I shows the coefficients a and b of the regression lines, along with the correlation coefficients, r, and the standard deviations of estimates of AY at the level of turbidity of 10 units, as calculated from the residual variance TABLE
I
CORRECTIONS
FOR
LIPEMIA
b
S(A
r
cl
YIO)*
% of AYIO
Chloride
(mmol/l)
Phosphate Iron
0.45
(mmol/l)
0.020
Creatinine
* S(A
0.402
8.0
0.981
0.0070
4.3 3.2
0.53
0.992
0.643
0.06
0.972
0.360
7.3
-5.10
3.25
0.990
1.39
2.6
1.95
(fimol/l)
0.987
-0.50
(firnOl/l)
Bilirubin
0.51 --a.035
(~mol/l)
Y IO) is the errw
of A Y at T = 10 units,
calculated
line. **
S(A
Ylo)
expressed
in percent
of AY
at T = 10 units.
from
the residual
error
around
**
the regression
TABLE
II
QUALITY
CON’I‘KOL
COMPAKISONS
Period M.D.’
Chloride
(mmol/l)
Phosphate Iron
(mnol/I)
(pmol/l)
Crrdtininr
1
Period li*
of the
M.D.
f
3
M.D.
1
1.52
n.s.
0.3
0.43
n.s.
I.7
1.69
11,s.
n.s.
0.02
1.42
11,s.
~0.04
1.23
11.s.
-0.4
I .03
n.s.
-0.2
0.54
11,s.
0.7
1.29
n.s.
0.82
11,s.
--2.8
1.38
11.s.
1.0
0.58
n.s.
I.2
?i measurements, not
Period
1.61
denotesdifference
n.s.,
2
-0.9
between
the rman
t = IS, -x_? l/j(( nl - 1)s: + (n2 ~ I)& tively.
METHODS
-0.02
(pmol/l)
(i M.D. * +
BETWEEN
significant
subscripts
“1”
at a 1, = 0.05
and
of
the
AutoChemist
and
the
01~+ ~I~)/(“~~I~)(I~~ + “2 “2”
refer
significance
to
the
AutoChemist
other 2).
method. where
and
other
i.e.
S’
-YI
s-2.
1s the variance
method.
respec-
level
around the regression line [ 151 . In the last column these standard deviations are related to AY, 0 (the AY at T = 10 units) and expressed as percentages. The errors of the corrections amount to 2----8% of the corrections, which is considered highly acceptable, e.g. an uncorrected value of 70 pmol/l of creatinine at a turbidity of 10 units will be corrected into 124 pmol/l with an error of i- 1 pmol/l (apart from the error involved in the measurement of the uncorrected 70 pmol/l). The significance of the intercept a is verified by the calculation of its error from the residual variance around the regression line. In all cases the 95% confidence interval around a was found to include the value zero; a is thus not significantly different from zero, as also predicted by theory (AY = 0 for T = 0). In the case of bilirubin and creatinine the correction is in the direction which increases the results, as the turbidity interference tends to elevate the blank values. Table II shows the mean differences between methods found from the internal quality control prograrn. Also given are the t values for three onemonth periods. The outcome of the t tests on the difference between AutoChemist and other methodologies was in all cases not statistically significant at the 95% confidence level. This supports the validity of the empirical relations in which AY is the difference between results found on the AutoChemist and from other methods, in that no significant bias exists between the basic measurement methods. The effect of the introduction of the correctives in routine use can be appraised from Fig. 1, which shows results from an external quality control programme. The graph is a cusum presentation of phosphate analysis. The ordinate represents the cusum of the percentage difference(s) between the value(s) of our laboratory and the reference value(s) for each weekly sample. The actual turbidity values in each sample are indicated on the graph; it is seen how high turbidity values coincide with jumps on the cusum curve. Obviously, the artifact of turbidity is removed as from the introduction of the correction.
11
of dtfferences
Curum
PHOSPHATE
Fig.
1.
Cusum
between sample
presentation
result(s) is
found
noted
on
of and
the
week
20
IO quality
reference
curve.
The
control
results
value(s) cusum
on phosphate
are summed curve
no
levels
on off
analyses.
the
Y-axis.
as from
the
The The
percentage turbidity
difference(s) in each
introduction
of
the
weekly
turbidity
correction.
Bilirubin corrections Table III shows in a similar manner to Table I the coefficients a, b, r for the case of bilirubinemia. From the residual variance around the regression line error estimates of AY are calculated at a bilirubin level of 80 pmol/l (approximately four times the upper limit of normals). These standard deviations and their percentages of AYs 0 (the AY value at B = 80 pmol/l) are given in the last two columns of Table III. Again the errors of AY are small (l--10%) and again a verification of the significance of a shows a to be not significantly different from zero, except in the case of alkaline phosphatase, where a significant intercept of 0.7 U/l was found. The random error given in Table III for alkaline phosphatase is extremely small (note r = 1.000); but in addition a systematic error component of 0.7 U/l is present. In view of the reference range for alkaline phosphatase of 29-88 U/l this bias is negligible. TABLE
III
CORRECTIONS
FOR
BILIRUBINEMIA
b
r
a
.s(A
Y&*
$2 of AY80
Chloride Iron
(mmolil)
(pmol/l)
Cholesterol Acid
(mmol/l)
phosphatase
Alkaline Uric
acid,
blank
Creatinine. LDH.
(U/l)
phosphatase
blank
blank
* * S(A
(mmol/I) (pmol/I)
0.988
0.167
0.41
0.999
0.160
0.0058
- 0.08
0.995
0.0389
10.1
0.0103
0.1
0.992
0.0576
3.5
0.036
0.7
1.000
0.0044
0.1
0.00141
0.009
0.998
0.0021~
1.8
0.9998
0.266
0.62
0.999
0.672
1.4
-1.05 1.30
0.58
is the
around Y~o).
0.009
0.107
0.509
(U/I)
* .5’(A Ygg) error
(U/l)
0.041
error
of
the regression
expressed
A Y at a bilimbin
concentration
of 80 pmol/l.
calculated
5.1 1.8
from
line.
as a percentage
of h Y at a bilirubin
concentration
of 80
**
umolil.
the residual
12
In the cases of uric acid, creatinine, and lactate dehydrogenase (LDH) the influence of bilirubin is compensated only in as much as the blank and main channels are equally elevated. The importance of using identical sensitivity settings for the two channels (factor converting signal to medical units) is stressed in this connection. Calibration sera often have very low blank values, the practise of compensating for the low blank signals by increasing the amplifier gain and attenuating the sensitivity factor is not recommendable in view of above interference considerations (optical interference). Discussion It is a unique asset of a multichannel system that all specimens treated can be measured for interfering substances such as e.g. bilirubin or turbidity. The availability of an on-line computer facilitates corrections for interferences. The integration of an on-line computer system in a conventional laboratory is naturally just as adequate for correction purposes provided the basic parameters are measured. The measurement of turbidity at 720 nm is chosen somewhat arbitrarily; however, it leads to an adequate empirical corrective on the tests shown in Table I. Turbidity in serum is caused by particles of varying diameter and concentration as discussed on a theoretical basis by Gautschi and Richterich [ 161, who also describe model experiments using latex suspensions to simulate turbidity. The use of computer corrections for lipemia, and bilirubinemia on the AutoChemist system was introduced by Jungner [17], however, the turbidity corrections were established differently. The influence of bilirubin on cholesterol in the direct LiebermannBurchard procedure was studied by van Boetzelaer and Zondag [18], who found that 1 mg/lOO ml of bilirubin produced the same absorbance as 5 mg/lOO ml of cholesterol measured at 615 nm on the Beckman DU spectrophotometer (equivalent to: b = 0.0076, mmol cholesterol/pmol bilirubin). The correction found in this paper for cholesterol measurements at 595 nm is somewhat smaller (b = 0.0058 * 0.0003). The application of a similar correction for haemolysis based on the haemoglobin concentration is not considered here. Such corrections are probably not recommendable [ 11, as the elevation of the measurement values for certain constituents (K, LDH, etc.) caused by haemolysis may not be an artifact of laboratory procedures but may well be biological. The errors of AY given in Tables I and III are based on the assumption that X in the relation AY = a + bX is found without error. Actually, X has an error, in that T or B are measurements. Error estimates obtained under such conditions tend to overestimate the true error of AY; however, predictions of A Y based on measured X values are valid, in that the random fluctuations of X will be equally present in the data used in the analysis and for prediction [ 191. The general problem discussed in this paper is focused on the individual patient specimen and will not be covered by the use of quality control sera for precision control.
13
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