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Application of cellulose acetate electrophoresis to globin chain separation for antenatal diagnosis of beta thalassemia
Clinics
Chimica
Acta,
Elsevier/North-Holland
CCA
137
116 (1981) 137- 142 Biomedical Press
1886
Application of cellulose acetate electrophoresis to globin chain separation for antenatal diagnosis of beta thalassemia M. Boccacci *, A. Massa and L. Tentori Istituto
Superiore
di Sanith
Luhoratorio
(Received
di Patologia
January
non Itzfettioa,
Roma (Itu!v)
14th. 1981)
Summary
Application of cellulose acetate electrophoresis to globin chain separation for antenatal diagnosis of beta thalassemia has been studied. A good correlation between an electrophoretic and a chromatographic method on carboxymethylcellulose was found and the diagnoses suggested by both methods were always coincident. The electrophoretic method was also utilized for HbS//&thalassemia diagnosis.
Introduction
Recently methods have been described for globin chain separation by cellulose acetate electrophoresis, using urea-phosphate buffers at slightly acid pH instead of ion exchange chromatography on carboxymethylcellulose [l-4]. The new method, applied to the study of the biosynthetic ratio of globin chains in reticulocytes from subjects who are carriers of the thalassemia variants or other blood disorders, gives results in good agreement with those obtained by the chromatographic method and appears particularly suitable as a diagnostic tool for large scale screening. In our laboratory, for the past year, we have performed the biochemical tests on fetal blood cells for antenatal diagnosis of homozygous thalassemia in association with the II Clinica Ostetrica e Ginecologica of the University of Rome. For chain separation and p/y biosynthetic ratio evaluation, we used a chromatographic method which is precise and accurate, but expensive and time-consuming; for that reason, we have studied and evaluated the use, for antenatal diagnosis, of the electrophoretic method which is cheaper and faster. Difficulties could arise from too low radioactivity levels since the /3/y biosynthetic ratio in homozygous fetuses is no more than 0.02, but when tests were *
To whom correspondence
0009~8981/81/0000-0000/$02.50
should be addressed. 0 Elsevier/North-Holland
Biomedical
Press
138
p/y
Chromatography
Fig. I. Regression line for the a/y ratios of 42 samples obtained by the electrophoretic and chromatographic methods. Regression equation: y = - 0.005 + 1.005x; scatter: Syx = t0.007; coefficient of correlation: r=0.971 (r2 =0.943); intercept: u= -0.005; slope: h= 1.005.
performed under our incubation conditions, we found radioactivity values so high that we could attain an accurate determination even in these cases. Therefore, utilizing samples with different concentrations of fetal and maternal cells relating to the homozygous, the heterozygous and the normal condition, we determined the biosynthetic ratio by the electrophoretic method, and compared the results with those obtained by the-chromatographic method. Statistical evaluation shows a good correlation between the two (Fig. 1). Materials and methods
Incubation of red blood cells with [ 3H]leucine, globin preparation and chain chromatographic separation were carried out as described by Alter et al. 151. Electrophoresis
Globin was dissolved to a concentration of 10 g/l in 0.04 mol/l sodium phosphate buffer, pH 6.3, containing 6 mol/l urea and 2% 2-mercaptoethanol. About 5 ~1 of globin solution were applied 3.5 cm from the anode end of a cellulose
139 30 i
a dps
p/y = 0.007 20
1c
0
5
10
15
20
25
30
mm
Fig. 2. Radioactivity values on single 1mm slices cut out from the strip after electrophoretic separation of the chains in a case of homozygous b-thalassemia.
acetate strip (Cellogel 250, Chemetron, Milan) previously equilibrated in phosphateurea buffer without 2-mercaptoethanol. Electrophoresis was carried out in the same buffer for 4 h at 140 V (constant voltage). Staining An aqueous solution was employed containing 5 g Ponceau-S and 100 g sulfosalicylic acid per liter. The strips were stained for 5 min and then washed several times with 5% acetic acid in distilled water. Quantitation of radioactive chains The stained bands, corresponding to the chains, were cut out and dissolved in 1 ml of a solution consisting of 88% diethylenedioxide, 10% methanol and 2% ethyleneglycol; this was mixed with 10 ml of liquid scintillation solution (Insta-Gel, Packard) and counted in a liquid scintillation counter with automated quenching correction and a counting efficiency of about 40%. Similar to the observation of Vettore et al. [2] with Coomassie blue, we found only a light quenching effect with Ponceau Red and we obtained absorbance and radioactivity values proportional to the globin quantity applied to the strip. To correct for background we cut out a strip between the alpha and beta bands, closer to the beta. We obtained stiIl better results measuring radioactivity on single 1 mm slices cut from the whole area including the chain bands (Fig. 2).
140
Results
In order to study the correlation between the electrophoretic and chromatographic methods, we determined the biosynthetic ratio on 42 samples with different concentrations of fetal and maternal cells and for which a range of p/y ratio from 0.07 to 0.150 had been previously found by the chromatographic method. The statistical evaluation of the data indicated a significant correlation between the two methods, as shown in Fig. 1, in which the regression line and related parameters are shown. In most cases, electrophoresis values were lo- 15% lower than the chromatographic ones. Such a difference could be due to an excess of radioactivity in the strip used for background correction. Better results were achieved by examining single 1 mm slices, as described in the methods section. A graph obtained in such a way is illustrated in Fig. 2, which is related to a sample with a very low /3/y ratio, not determinable with the usual correction, because the background value was greater than the value of the beta band. A p/y ratio = 0.007 was obtained in good agreement with that obtained by the chromatographic method (= 0.008). When a high degree of accuracy was required, the latter procedure is certainly advisable, even if it involves radioactivity determination in 30-40 samples instead of the usual four.
TABLE
I
RESULTS OBTAINED BY THE ELECTROPHORETIC, BY THE CHROMATOGRAPHIC METHOD
Nor, normal;
WITH
THOSE
Diagnosis
/3/y ratio
M.C. C.C. B.M.T. C.D. L.D. M.R. R.C. M.A. S.R. I.M. B.L. O.F. P.C. C.M.T. R.M. T.G. B.A. C.R. M.M. R.A.
COMPARED
electr.
chrom.
electr.
chrom.
0.01 I 0.054 0.041 0.043 0.076 0.005 0.041 0.040 0.005 0.100 0.040 0.046 0.005 0.041 0.008 0.061 0.040 0.078 0.000 0.076
0.022 0.054 0.036 0.041 0.087 0.020 0.049 0.050 0.009 0.100 0.042 0.052 0.012 0.043 0.005 0.070 0.036 0.075 0.000 0.085
Horn Het Het Het Nor Horn Het Het Horn Nor Het Het Horn Het Horn Het Het Nor Horn Nor
Horn Het Het Het Nor Horn Het Het Horn Nor Het Het Horn Het Horn Het Het Nor Horn Nor
Het, heterozygous;
Horn, homozygous.
OBTAINED
141
+
Fig. 3. Electrophoretic
separation
of the globin chains in a case of HbS/P-thalassemia.
To establish the validity of the electrophoretic method, in the second part of our study we utilized this technique for antenatal diagnosis of /?-thalassemia in 20 cases. Each result was then checked using the chromatographic method and the results are illustrated in Table I, from which it is apparent that they were always coincident. pO-Thalassemia results in no chain production by the fetus; p + -thaIassemia is a more variable condition, but in any case the /3/y ratio is lower than 0.030. The /3/y ratio for fetuses with the thalassemia trait ranges from 0.036 to 0.070 and that for normals from 0.070 to 0.150. A fetus at risk for HbS//3-thalassemia was examined, too; Fig. 3 shows the chain separation and demonstrates that electrophoresis provides good resolution, also for the /3” band: the values of band ratios agree with those obtained by chromatography. Discussion
The results obtained allow us to conclude that the electrophoretic method for chain separation and p/y biosynthetic, ratio evaluation appears remarkably useful for antenatal diagnosis of homozygous P-thalassemia, due to its rapidity and simplicity. The examination of samples performed by ehectrophoresis can provide information quickly and so assist in the management of cases, and the planning of further investigation; moreover, the method offers a means of comparison of values, very useful for resolution of the most difficult cases, as when the resulting value is placed at the cut-off point between /I + -homozygosis and heterozygosis. Acknowledgement
This work was partially supported ventiva” (Contract No. 80.01171.83).
by a CNR grant, project “Medicina
Pre-
142
References 1 Salmon, J.E., Schihrb, G., Natta, L.N. and Bank, A. (1978) Q uantitation of human globin chain synthesis by cellulose acetate electrophoresis. Anal. Biochem. 91, 146- I57 2 Vettore, LT, De Matteis, M.C., Bassetto, M.A. and Pepe, G.M. (1978) Biosynthetic ratio of labelled globin chains in human reticulocytes determined by electrophoresis on cellulose acetate. Hemoglobin 2, l29- I41 of globin chains using cellulose acetate 3 Barton, J., Smith, M.B. and Cauchi, M.N. (1978) Q uantitation electrophoresis: analysis of fetal globin chain production. Am. J. Hematol. 5, 341-345 4 Harano, T., Ueda, S., Harano, K. and Shibata, S. (1980) Improved method for quantitation of biosynthesized human globin chains in reticulocytes by use of urea cellulose acetate membrane electrophoresis. Proc. Japan, Acad. 56, Ser. B, 230-234 5 Alter, B.P., Modell, B., Fairweather, D., Hobbins, J.C., Mahoney, and Nathan, D.G. (1976) Prenatal diagnosis of hemoglobinopathies.
M.J., Frigoletto, F.D., Sherman, New Engl. J. Med. 295. l437-
A.S. 1443
Chimica
Acta,
Elsevier/North-Holland
CCA
137
116 (1981) 137- 142 Biomedical Press
1886
Application of cellulose acetate electrophoresis to globin chain separation for antenatal diagnosis of beta thalassemia M. Boccacci *, A. Massa and L. Tentori Istituto
Superiore
di Sanith
Luhoratorio
(Received
di Patologia
January
non Itzfettioa,
Roma (Itu!v)
14th. 1981)
Summary
Application of cellulose acetate electrophoresis to globin chain separation for antenatal diagnosis of beta thalassemia has been studied. A good correlation between an electrophoretic and a chromatographic method on carboxymethylcellulose was found and the diagnoses suggested by both methods were always coincident. The electrophoretic method was also utilized for HbS//&thalassemia diagnosis.
Introduction
Recently methods have been described for globin chain separation by cellulose acetate electrophoresis, using urea-phosphate buffers at slightly acid pH instead of ion exchange chromatography on carboxymethylcellulose [l-4]. The new method, applied to the study of the biosynthetic ratio of globin chains in reticulocytes from subjects who are carriers of the thalassemia variants or other blood disorders, gives results in good agreement with those obtained by the chromatographic method and appears particularly suitable as a diagnostic tool for large scale screening. In our laboratory, for the past year, we have performed the biochemical tests on fetal blood cells for antenatal diagnosis of homozygous thalassemia in association with the II Clinica Ostetrica e Ginecologica of the University of Rome. For chain separation and p/y biosynthetic ratio evaluation, we used a chromatographic method which is precise and accurate, but expensive and time-consuming; for that reason, we have studied and evaluated the use, for antenatal diagnosis, of the electrophoretic method which is cheaper and faster. Difficulties could arise from too low radioactivity levels since the /3/y biosynthetic ratio in homozygous fetuses is no more than 0.02, but when tests were *
To whom correspondence
0009~8981/81/0000-0000/$02.50
should be addressed. 0 Elsevier/North-Holland
Biomedical
Press
138
p/y
Chromatography
Fig. I. Regression line for the a/y ratios of 42 samples obtained by the electrophoretic and chromatographic methods. Regression equation: y = - 0.005 + 1.005x; scatter: Syx = t0.007; coefficient of correlation: r=0.971 (r2 =0.943); intercept: u= -0.005; slope: h= 1.005.
performed under our incubation conditions, we found radioactivity values so high that we could attain an accurate determination even in these cases. Therefore, utilizing samples with different concentrations of fetal and maternal cells relating to the homozygous, the heterozygous and the normal condition, we determined the biosynthetic ratio by the electrophoretic method, and compared the results with those obtained by the-chromatographic method. Statistical evaluation shows a good correlation between the two (Fig. 1). Materials and methods
Incubation of red blood cells with [ 3H]leucine, globin preparation and chain chromatographic separation were carried out as described by Alter et al. 151. Electrophoresis
Globin was dissolved to a concentration of 10 g/l in 0.04 mol/l sodium phosphate buffer, pH 6.3, containing 6 mol/l urea and 2% 2-mercaptoethanol. About 5 ~1 of globin solution were applied 3.5 cm from the anode end of a cellulose
139 30 i
a dps
p/y = 0.007 20
1c
0
5
10
15
20
25
30
mm
Fig. 2. Radioactivity values on single 1mm slices cut out from the strip after electrophoretic separation of the chains in a case of homozygous b-thalassemia.
acetate strip (Cellogel 250, Chemetron, Milan) previously equilibrated in phosphateurea buffer without 2-mercaptoethanol. Electrophoresis was carried out in the same buffer for 4 h at 140 V (constant voltage). Staining An aqueous solution was employed containing 5 g Ponceau-S and 100 g sulfosalicylic acid per liter. The strips were stained for 5 min and then washed several times with 5% acetic acid in distilled water. Quantitation of radioactive chains The stained bands, corresponding to the chains, were cut out and dissolved in 1 ml of a solution consisting of 88% diethylenedioxide, 10% methanol and 2% ethyleneglycol; this was mixed with 10 ml of liquid scintillation solution (Insta-Gel, Packard) and counted in a liquid scintillation counter with automated quenching correction and a counting efficiency of about 40%. Similar to the observation of Vettore et al. [2] with Coomassie blue, we found only a light quenching effect with Ponceau Red and we obtained absorbance and radioactivity values proportional to the globin quantity applied to the strip. To correct for background we cut out a strip between the alpha and beta bands, closer to the beta. We obtained stiIl better results measuring radioactivity on single 1 mm slices cut from the whole area including the chain bands (Fig. 2).
140
Results
In order to study the correlation between the electrophoretic and chromatographic methods, we determined the biosynthetic ratio on 42 samples with different concentrations of fetal and maternal cells and for which a range of p/y ratio from 0.07 to 0.150 had been previously found by the chromatographic method. The statistical evaluation of the data indicated a significant correlation between the two methods, as shown in Fig. 1, in which the regression line and related parameters are shown. In most cases, electrophoresis values were lo- 15% lower than the chromatographic ones. Such a difference could be due to an excess of radioactivity in the strip used for background correction. Better results were achieved by examining single 1 mm slices, as described in the methods section. A graph obtained in such a way is illustrated in Fig. 2, which is related to a sample with a very low /3/y ratio, not determinable with the usual correction, because the background value was greater than the value of the beta band. A p/y ratio = 0.007 was obtained in good agreement with that obtained by the chromatographic method (= 0.008). When a high degree of accuracy was required, the latter procedure is certainly advisable, even if it involves radioactivity determination in 30-40 samples instead of the usual four.
TABLE
I
RESULTS OBTAINED BY THE ELECTROPHORETIC, BY THE CHROMATOGRAPHIC METHOD
Nor, normal;
WITH
THOSE
Diagnosis
/3/y ratio
M.C. C.C. B.M.T. C.D. L.D. M.R. R.C. M.A. S.R. I.M. B.L. O.F. P.C. C.M.T. R.M. T.G. B.A. C.R. M.M. R.A.
COMPARED
electr.
chrom.
electr.
chrom.
0.01 I 0.054 0.041 0.043 0.076 0.005 0.041 0.040 0.005 0.100 0.040 0.046 0.005 0.041 0.008 0.061 0.040 0.078 0.000 0.076
0.022 0.054 0.036 0.041 0.087 0.020 0.049 0.050 0.009 0.100 0.042 0.052 0.012 0.043 0.005 0.070 0.036 0.075 0.000 0.085
Horn Het Het Het Nor Horn Het Het Horn Nor Het Het Horn Het Horn Het Het Nor Horn Nor
Horn Het Het Het Nor Horn Het Het Horn Nor Het Het Horn Het Horn Het Het Nor Horn Nor
Het, heterozygous;
Horn, homozygous.
OBTAINED
141
+
Fig. 3. Electrophoretic
separation
of the globin chains in a case of HbS/P-thalassemia.
To establish the validity of the electrophoretic method, in the second part of our study we utilized this technique for antenatal diagnosis of /?-thalassemia in 20 cases. Each result was then checked using the chromatographic method and the results are illustrated in Table I, from which it is apparent that they were always coincident. pO-Thalassemia results in no chain production by the fetus; p + -thaIassemia is a more variable condition, but in any case the /3/y ratio is lower than 0.030. The /3/y ratio for fetuses with the thalassemia trait ranges from 0.036 to 0.070 and that for normals from 0.070 to 0.150. A fetus at risk for HbS//3-thalassemia was examined, too; Fig. 3 shows the chain separation and demonstrates that electrophoresis provides good resolution, also for the /3” band: the values of band ratios agree with those obtained by chromatography. Discussion
The results obtained allow us to conclude that the electrophoretic method for chain separation and p/y biosynthetic, ratio evaluation appears remarkably useful for antenatal diagnosis of homozygous P-thalassemia, due to its rapidity and simplicity. The examination of samples performed by ehectrophoresis can provide information quickly and so assist in the management of cases, and the planning of further investigation; moreover, the method offers a means of comparison of values, very useful for resolution of the most difficult cases, as when the resulting value is placed at the cut-off point between /I + -homozygosis and heterozygosis. Acknowledgement
This work was partially supported ventiva” (Contract No. 80.01171.83).
by a CNR grant, project “Medicina
Pre-
142
References 1 Salmon, J.E., Schihrb, G., Natta, L.N. and Bank, A. (1978) Q uantitation of human globin chain synthesis by cellulose acetate electrophoresis. Anal. Biochem. 91, 146- I57 2 Vettore, LT, De Matteis, M.C., Bassetto, M.A. and Pepe, G.M. (1978) Biosynthetic ratio of labelled globin chains in human reticulocytes determined by electrophoresis on cellulose acetate. Hemoglobin 2, l29- I41 of globin chains using cellulose acetate 3 Barton, J., Smith, M.B. and Cauchi, M.N. (1978) Q uantitation electrophoresis: analysis of fetal globin chain production. Am. J. Hematol. 5, 341-345 4 Harano, T., Ueda, S., Harano, K. and Shibata, S. (1980) Improved method for quantitation of biosynthesized human globin chains in reticulocytes by use of urea cellulose acetate membrane electrophoresis. Proc. Japan, Acad. 56, Ser. B, 230-234 5 Alter, B.P., Modell, B., Fairweather, D., Hobbins, J.C., Mahoney, and Nathan, D.G. (1976) Prenatal diagnosis of hemoglobinopathies.
M.J., Frigoletto, F.D., Sherman, New Engl. J. Med. 295. l437-
A.S. 1443