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Assessment of a simple electrophoretic method for measuring HbA1
487
Clinica
Chimica
Acta,
108 (1980)
@ Elsevier/North-Holland
487-491 Biomedical Press
BRIEF TECHNICAL NOTE CCA 1576
ASSESSMENT OF A SIMPLE ELECTROPHORETIC METHOD FOR MEASURING HbAl
ANN READ, LAILA TIBI and A.F. SMITH * University (U.K.)
Department
of Clinical
Chemistry,
The Royal
Infirmary,
Edinburgh
EH3
9YW
(Received June 16th, 1980)
Measurements of glycosylated haemoglobin (HbA1) are finding increased use in the management of patients with diabetes mellitus. However, present analytical methods are either tedious or prone to error. The commonest method uses small ion-exchange columns and is fairly simple, but the results depend greatly on analytical and environmental variables such as differences between batches of ion-exchangers, reaction pH and reaction temperature [ 11. Failure to recognise these problems, due to unavailability of adequate quality control and standard material, may lead to grossly inaccurate results. We have assessed a simple, commercially available method of separating HbAl electrophoretically on agar gel films. Methods Samples Samples of heparinised blood were obtained from healthy individuals and from diabetics. The latter group included both poorly and well controlled patients, children and closely controlled pregnant women. Electrophore tic method This is based on a widely used electrophoresis system (Corning Medical, Halstead, U.K.). The novel feature of the present method is claimed to lie in the formulation of the agar used on the plastic-backed electrophoresis films, which allows the separation of HbAl from the main Hb fraction. Blood was diluted 1 : 4 in haemolysing solution (containing 0.1% saponin and 0.05% EDTA in distilled water) and mixed by vortex; if electrophoresis was not performed immediately, the haemolysate was’stored at 4°C. * To whom correspondence should be directed.
488
1 kl haemolysate was applied to the special agar film in the preformed sample slot. Electrophoresis was performed at 60 V constant voltage for 35 min, the strip removed from the tank, dried at 6O”C, then scanned on a densitometer (Coming 720). The percentage of HbAl was obtained from the area under the corresponding peak. Ion-exchange
chromatography
Small ion-exchange columns (6.0 X 0.7 cm) were used to separate HbAl from HbA [2]. We found it necessary to modify the published method by preequilibrating the resin with several changes of buffer over 2-3 weeks before the resin pH remained stable at pH 6.75. In our hands this method correlated well with other ion exchange methods [3] and, over a period of several months had a between-batch C.V., calculated from repeat assays of specimens from patients, of 3.5%. Results The electrophoretic method was very simple to perform and no difficulties were experienced even by staff unfamiliar with the technique or apparatus. HbAl was well separated from the main Hb component (Fig. 1). Accuracy
In the absence of any reference method for determining HbAl, we have compared the results with those obtained using the ion-exchange method (Fig. 2). In general, there is good agreement and scatter around the line of best fit can largely be attributed to the imprecision of the two methods. However, the
Fig. 1. Densitometric scan of separated Hb fractions, showing main Hb fraction on the left and HbAl the right.
on
489
HbA,
14.0
EIectrophoresis
12.0
10.0
6.0
6.0
4.0
2.0
0.0
/ I
0.0
I’
I
I
I
I
I
I
I
I
1
2.0
a.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
HbAl
-
ion-exchange
1 20.0
column
Fig. 2. Correlation between HbAl (expressed as a percentage of total Hb) measured by the electrophoretic and ion-exchange methods. Y = -2.7 + 1.29x, P = 0.95. SEA 0.37, SEB 0.036. regression line fitted on assumption both X and Y subject to error [ 41.
slope of the regression line is si~ificantly different from 1.0 and the intercept is significantly removed from the origin. Within the reference range, these effects are largely self-cancelling but at higher HbAl levels the electrophoretic method gives higher results. Precision Only between-batch, between-day precision was assessed since preliminary assessment showed good within-batch (within film) precision (C.V. < 3%). For a pooled haemolysate (mean HbAl 11.3%) the S.D. was 0.54% (C.V. 4.7%) -total number of analyses 19. For repeated analysis on specimens from patients (mean HbAl 9.7%, range 4.6%-17.5%) the SD. was 0.61% (C.V. 6.3%) - total number of replicate analyses 25. Repeat scanning of films showed a C.V. of less than 1.5%, indicating that most of the imprecision arose during specimen processing, electrophoresis and drying of films.
490
Effect
of sample storage
The haemolysates from six samples with HbAl ranging from 6% to 12% were analysed every day for one week. None of the samples showed a significant change in HbA1, nor was any trend apparent in the group as a whole. Reference
range
Although no formal studies were performed, HbA, levels were measured in 22 healthy adults (10 male, 12 female), aged between 20 and 50, in whom there was no clinical evidence of diabetes or anaemia. The results were approximately normally distributed with mean 6.3% (S.D. 0.8%). The resulting 95% range of 4.7-7.9% was close to our laboratory reference range for the column method of 5.0-7.6%.
Conclusions We have shown that the commercially available method of separating HbAl by electrophoresis and subsequent densitometric scanning gives results which correlate well with our column chromatographic technique. The fact that the regression line for the comparison does not have unit slope nor go through the origin seems to us to be due, in all probability, to inaccuracy of the column procedure, which is extremely susceptible to temperature variation. Although we routinely “correct” for this variation so that our long-term precision for the method is good, it is difficult to be certain that we are correcting to the true value. The electrophoretic method is simple and quick and requires little technical skill. The chromatographic method, if it is to perform reliably, requires great attention to the preparation of the buffered resin and to avoidance of, or correction for, temperature variations. Furthermore, they use cyanide in the buffers used for elution of the Hb. This reagent should be avoided if at all possible, especially in methods which will be used routinely. In our experience, the commercially available mini-columns, which are quite quick to use, have problems with day-today and batch-to-batch variation. We have also used the calorimetric method [5] but found it to compare less well with the chromatographic, and also to be rather tedious and time-consuming. The penalty one pays in using the electrophoretic method is that the precision is less good than that usually obtainable using chromatographic methods. It is for individual laboratories, and the clinicians they serve, to decide whether the convenience and simplicity of the electrophoretic method offset this loss of precision. Nevertheless, it is our impression, based on personal observations, that this degree of imprecision is very small when compared to discrepancies between laboratories. Acknowledgements We would like to thank Corning Medical for the supply of strips and Mrs. Brenda Shaw for skilled technical assistance.
491
References 1 Dix, D., Cohen, P., Kingsley, S.. Senkbeil, J. and Sexton, K. (1978) Evaluation of a glyco~emo~obin kit. Clin. Chem. 24,2073 2 Welch, S.G. and Boucher, B.J. (1978) A rapid micro-scale method for the measurement of Haemoglobin Al~a+b+~). Diabetologia 14, 209-211 3 Kynoch, P.A.M. and Lehmann, H. (1977) Rapid estimation (2% h) of glycosylated haemoglobins for routine purposes. Lancet ii, 16 4 Wakkers, P.J.M., HeIlendoorn. H.B.A., Op de Weegh, G.J. and Heexspink, W. (1975) Applications of statistics in clinical chemistry: a critical evaluation of regression lines. Clin. Chim. Acta 64, 173-184 5 Fluckiger, R. and Winterhalter, K.H. (1976) In vitro synthesis of haemoglobin Ale. FEBS Lett. 71,356
~eon~.~ued
from
couer)
Short Communications Methylthioadenosine nucleosidase in normal and dystrophic human muscle N.C. Kar and C.M. Pearson (Los Angeles, CA, U.S.A.) . . . , . , . , . . . . . . . . . . . . Altered erythrocyte spectrin extractability in Duchenne muscular dystrophy Y. Nagano, P. Wong and A.D. Roses (Durham, NC, U.S.A.) . . . . . . . . . . . . . . . . Prenatal detection of defects in propionate metabolism Il. Wendel (Diisseldorf, F.R.G.) . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
465 469 4’75
Brief Technical Notes Rapid and efficient extraction of steroids from microfitre quantities of plasma using E~trelut~in~-columns E. Bamberg, E. Mostl, N. El Din Hassaan, W. Stock1 and H.S. Choi (Vienna, Austria) Determination de l’activith de la transc&olase drythrocytaire E. Delacoux, J. Sancho, Th. Evstigneeff, L. Coulombier and S. Langin (Paris, France)................................................... Assessment of a simple electrophoretic method for measuring HbAi A. Read, L. Tibi and A.F. Smith (Edinburgh, U.K.) . . . . . . . . . . . . . . . . . . . . . . Enzymatic assay of serum sialic acid K. Sugahara, K. Sugimoto, 0. Nomura and T. Usui (Nagasaki, Japan) . . . . . . . . . .
479
483 487 493
International Federation of Clinical Chemistry IFCC (1980) Section No. 4 ....................................... .4991 Physicochemical Quantities and Units in Clinical Chemistry with special emphasis ............................... on activities and activity coefficients 5OlF Author Index .................................................
540
Subject Index
545
................................................
Errata ......................................................
553
Clinica
Chimica
Acta,
108 (1980)
@ Elsevier/North-Holland
487-491 Biomedical Press
BRIEF TECHNICAL NOTE CCA 1576
ASSESSMENT OF A SIMPLE ELECTROPHORETIC METHOD FOR MEASURING HbAl
ANN READ, LAILA TIBI and A.F. SMITH * University (U.K.)
Department
of Clinical
Chemistry,
The Royal
Infirmary,
Edinburgh
EH3
9YW
(Received June 16th, 1980)
Measurements of glycosylated haemoglobin (HbA1) are finding increased use in the management of patients with diabetes mellitus. However, present analytical methods are either tedious or prone to error. The commonest method uses small ion-exchange columns and is fairly simple, but the results depend greatly on analytical and environmental variables such as differences between batches of ion-exchangers, reaction pH and reaction temperature [ 11. Failure to recognise these problems, due to unavailability of adequate quality control and standard material, may lead to grossly inaccurate results. We have assessed a simple, commercially available method of separating HbAl electrophoretically on agar gel films. Methods Samples Samples of heparinised blood were obtained from healthy individuals and from diabetics. The latter group included both poorly and well controlled patients, children and closely controlled pregnant women. Electrophore tic method This is based on a widely used electrophoresis system (Corning Medical, Halstead, U.K.). The novel feature of the present method is claimed to lie in the formulation of the agar used on the plastic-backed electrophoresis films, which allows the separation of HbAl from the main Hb fraction. Blood was diluted 1 : 4 in haemolysing solution (containing 0.1% saponin and 0.05% EDTA in distilled water) and mixed by vortex; if electrophoresis was not performed immediately, the haemolysate was’stored at 4°C. * To whom correspondence should be directed.
488
1 kl haemolysate was applied to the special agar film in the preformed sample slot. Electrophoresis was performed at 60 V constant voltage for 35 min, the strip removed from the tank, dried at 6O”C, then scanned on a densitometer (Coming 720). The percentage of HbAl was obtained from the area under the corresponding peak. Ion-exchange
chromatography
Small ion-exchange columns (6.0 X 0.7 cm) were used to separate HbAl from HbA [2]. We found it necessary to modify the published method by preequilibrating the resin with several changes of buffer over 2-3 weeks before the resin pH remained stable at pH 6.75. In our hands this method correlated well with other ion exchange methods [3] and, over a period of several months had a between-batch C.V., calculated from repeat assays of specimens from patients, of 3.5%. Results The electrophoretic method was very simple to perform and no difficulties were experienced even by staff unfamiliar with the technique or apparatus. HbAl was well separated from the main Hb component (Fig. 1). Accuracy
In the absence of any reference method for determining HbAl, we have compared the results with those obtained using the ion-exchange method (Fig. 2). In general, there is good agreement and scatter around the line of best fit can largely be attributed to the imprecision of the two methods. However, the
Fig. 1. Densitometric scan of separated Hb fractions, showing main Hb fraction on the left and HbAl the right.
on
489
HbA,
14.0
EIectrophoresis
12.0
10.0
6.0
6.0
4.0
2.0
0.0
/ I
0.0
I’
I
I
I
I
I
I
I
I
1
2.0
a.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
HbAl
-
ion-exchange
1 20.0
column
Fig. 2. Correlation between HbAl (expressed as a percentage of total Hb) measured by the electrophoretic and ion-exchange methods. Y = -2.7 + 1.29x, P = 0.95. SEA 0.37, SEB 0.036. regression line fitted on assumption both X and Y subject to error [ 41.
slope of the regression line is si~ificantly different from 1.0 and the intercept is significantly removed from the origin. Within the reference range, these effects are largely self-cancelling but at higher HbAl levels the electrophoretic method gives higher results. Precision Only between-batch, between-day precision was assessed since preliminary assessment showed good within-batch (within film) precision (C.V. < 3%). For a pooled haemolysate (mean HbAl 11.3%) the S.D. was 0.54% (C.V. 4.7%) -total number of analyses 19. For repeated analysis on specimens from patients (mean HbAl 9.7%, range 4.6%-17.5%) the SD. was 0.61% (C.V. 6.3%) - total number of replicate analyses 25. Repeat scanning of films showed a C.V. of less than 1.5%, indicating that most of the imprecision arose during specimen processing, electrophoresis and drying of films.
490
Effect
of sample storage
The haemolysates from six samples with HbAl ranging from 6% to 12% were analysed every day for one week. None of the samples showed a significant change in HbA1, nor was any trend apparent in the group as a whole. Reference
range
Although no formal studies were performed, HbA, levels were measured in 22 healthy adults (10 male, 12 female), aged between 20 and 50, in whom there was no clinical evidence of diabetes or anaemia. The results were approximately normally distributed with mean 6.3% (S.D. 0.8%). The resulting 95% range of 4.7-7.9% was close to our laboratory reference range for the column method of 5.0-7.6%.
Conclusions We have shown that the commercially available method of separating HbAl by electrophoresis and subsequent densitometric scanning gives results which correlate well with our column chromatographic technique. The fact that the regression line for the comparison does not have unit slope nor go through the origin seems to us to be due, in all probability, to inaccuracy of the column procedure, which is extremely susceptible to temperature variation. Although we routinely “correct” for this variation so that our long-term precision for the method is good, it is difficult to be certain that we are correcting to the true value. The electrophoretic method is simple and quick and requires little technical skill. The chromatographic method, if it is to perform reliably, requires great attention to the preparation of the buffered resin and to avoidance of, or correction for, temperature variations. Furthermore, they use cyanide in the buffers used for elution of the Hb. This reagent should be avoided if at all possible, especially in methods which will be used routinely. In our experience, the commercially available mini-columns, which are quite quick to use, have problems with day-today and batch-to-batch variation. We have also used the calorimetric method [5] but found it to compare less well with the chromatographic, and also to be rather tedious and time-consuming. The penalty one pays in using the electrophoretic method is that the precision is less good than that usually obtainable using chromatographic methods. It is for individual laboratories, and the clinicians they serve, to decide whether the convenience and simplicity of the electrophoretic method offset this loss of precision. Nevertheless, it is our impression, based on personal observations, that this degree of imprecision is very small when compared to discrepancies between laboratories. Acknowledgements We would like to thank Corning Medical for the supply of strips and Mrs. Brenda Shaw for skilled technical assistance.
491
References 1 Dix, D., Cohen, P., Kingsley, S.. Senkbeil, J. and Sexton, K. (1978) Evaluation of a glyco~emo~obin kit. Clin. Chem. 24,2073 2 Welch, S.G. and Boucher, B.J. (1978) A rapid micro-scale method for the measurement of Haemoglobin Al~a+b+~). Diabetologia 14, 209-211 3 Kynoch, P.A.M. and Lehmann, H. (1977) Rapid estimation (2% h) of glycosylated haemoglobins for routine purposes. Lancet ii, 16 4 Wakkers, P.J.M., HeIlendoorn. H.B.A., Op de Weegh, G.J. and Heexspink, W. (1975) Applications of statistics in clinical chemistry: a critical evaluation of regression lines. Clin. Chim. Acta 64, 173-184 5 Fluckiger, R. and Winterhalter, K.H. (1976) In vitro synthesis of haemoglobin Ale. FEBS Lett. 71,356
~eon~.~ued
from
couer)
Short Communications Methylthioadenosine nucleosidase in normal and dystrophic human muscle N.C. Kar and C.M. Pearson (Los Angeles, CA, U.S.A.) . . . , . , . , . . . . . . . . . . . . Altered erythrocyte spectrin extractability in Duchenne muscular dystrophy Y. Nagano, P. Wong and A.D. Roses (Durham, NC, U.S.A.) . . . . . . . . . . . . . . . . Prenatal detection of defects in propionate metabolism Il. Wendel (Diisseldorf, F.R.G.) . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
465 469 4’75
Brief Technical Notes Rapid and efficient extraction of steroids from microfitre quantities of plasma using E~trelut~in~-columns E. Bamberg, E. Mostl, N. El Din Hassaan, W. Stock1 and H.S. Choi (Vienna, Austria) Determination de l’activith de la transc&olase drythrocytaire E. Delacoux, J. Sancho, Th. Evstigneeff, L. Coulombier and S. Langin (Paris, France)................................................... Assessment of a simple electrophoretic method for measuring HbAi A. Read, L. Tibi and A.F. Smith (Edinburgh, U.K.) . . . . . . . . . . . . . . . . . . . . . . Enzymatic assay of serum sialic acid K. Sugahara, K. Sugimoto, 0. Nomura and T. Usui (Nagasaki, Japan) . . . . . . . . . .
479
483 487 493
International Federation of Clinical Chemistry IFCC (1980) Section No. 4 ....................................... .4991 Physicochemical Quantities and Units in Clinical Chemistry with special emphasis ............................... on activities and activity coefficients 5OlF Author Index .................................................
540
Subject Index
545
................................................
Errata ......................................................
553