American Journal of Hematology 5:341-345 (1978)

Quantitation of Globin Chains Using Cellulose Acetate Electrophoresis: Analysis of Fetal Globin Chain Production Jennifer Barton, Merran B. Smith, and M.N. Cauchi

Section of Hematology, The Royal Women’s Hospital, Melbourne, Australia

A technique involving the quantitative estimation of globin chains using cellu- lose acetate electrophoresis is described, and applied to the analysis of fetal globin chain production. When cord blood was analyzed, there was poor corre- lation between the globin chain ratios namely P/a, y/a and P/y ratios with either age or weight of newborn babies between 36-40 weeks gestation: best correla- tions were obtained for the ratio against weight (P < 0.05). However, there was highly significant correlation between the various globin chain ratios and either age or weight of the newborn infant up to 12 weeks of age (P < 0.001). This technique allows visual demonstration and quantitation of abnormal hae- moglobins in a single procedure.

Key words: globin chains, fetal hemoglobin, electrophoresis

INTRODUCTION

A number of techniques have been developed for determining fetal hemoglobin, the ones most commonly used being the alkali denaturation technique, or electrophoresis using cellulose acetate or other supporting media. Schneider [ I ] developed a technique of electrophoresis of globin chains of hemoglobin using cellulose acetate in a buffer con- nique of electrophoresis of globin chains of hemoglobin using cellulose acetate in a buffer con- taining urea and 2-mercaptoethanol. In the following communication we describe the appli- cation of this technique for the quantitation of fetal globin chains in fetuses, cord blood, and newborn infants up to 12 weeks of age.

Received for publication April 21, 1978; accepted September 27,1978. Address reprint requests to Dr. M.N. Cauchi, Section of Hematology, Royal Women’s Hospital, Swanston Street, Carlton, 3053, Victoria, Australia.

0361-8609/78/0504-0341$01.40 0 1978 Alan R. Liss, Inc.

342 Barton, Smith, and Cauchi

MATERIAL AND METHODS

Blood samples were obtained from a) fetuses delivered prematurely, b) cord blood

Specimens of blood in heparin were washed twice in 9% NaCl, and 1 in 4 hemolysates from normal babies, and c) babies up to 12 weeks of age.

were prepared in distilled water. The red cell stroma was removed by centrifugation, and specimens were examined immediately or frozen at -20°C. The samples were then treated as described by Schneider (1). To 20 p1 of hemolysate were added 20 p1 of mercaptoethanol and 20 p1 of Tris EDTA boric acid urea buffer (Tris 2.4 gm, EDTA 0.1 gm, boric acid 0.7 gm, urea 360 gm per liter; pH adjusted with concentrated citric acid ti 6.0; and 5 ml of 2-mercapto- ethanal per liter were added to the buffer immediately prior to use). The mixture was allowed to stand for 30 minutes and then 0.5 p1 of hemolysate was applied to strips of Sepraphore 111 cellulose acetate (previously presoaked in the buffer) using a Beckman Microzone appli- cator. Electrophoresis was carried out in a Beckman Microzone tank for up to 1 hour at 300 volts using a buffer containing Tris 7.1 gm, EDTA 0.4 gm, boric acid 2.2 gin, and urea 360 gm/l: pH 6.0.2-mercaptoethanol was added to the buffer prior to use as described above. The specimens were stained with Ponceau S, washed in two percent acetic acid, cleared in dekalin and scanned in a Beckman Microzone R-110 densitometer.

RESULTS

Separation of globin chains on cellulose acetate electrophoresis was carried out and Figure 1 shows the densitometric scan from these preparations. There was a clear separation of the a, 0, P s , and y chains which could be measured quantitatively by’assessment of the area under the curve. The best results were obtained when electrophoresis was carried out for the minimum time required for adequate separation of the bands: this was normally 45-60 minutes. With longer times there was considerable loss of the Q! chain front, resulting in unreliable

60 i 10

d

Fig. 1. Densitornetric scan of globin chains following cellulose acetate electrophoresis of A) normal cord blood, B) cord blood from an infant with sickle cell trait, C) normal adult blood, and D) blood from an adult with sickle cell trait.

Quantitative Globin Electrophoresis 343

ratios. When artificial mixtures of normal adult and normal cord blood were examined by this technique (Fig. 2) a linear relationship was obtained between the ratio and the percentage of adult blood in the mixture. It is to be noted that levels of a globin below five percent cannot be detected by this technique.

This technique was applied to the analysis of globin chain ratios from samples of blood taken from 1) normal cord blood taken from the placenta immediately after birth, and b) blood taken from newborn infants to 12 weeks of age. Only densitometric scans yield- ing a ratio of p + y = 1 t 0.1 were included in this study. __

(Y

Figure 3 shows that there was minimal change in the globin chain ratios of cord blood taken from pregnancies of varying length of gestation. However, significant correlations

10 20 30 40 50 60 ioeb 90 l o o HbA

Fig. 2. chain ratios obtained from densitometric scans of artificial mixtures of adult and fetal blood.

Gestation (weeks) Weight (Gms)

Fig. 3. Globin chian ratios obtained from densitometric scans of cord blood plotted vs (a) age in weeks or (b) weight in grams.

344 Barton, Smith, and Cauchi

TABLE I. Correlation Between Globin Chain Ratios and a) Age or b) Weight in Cord Blood and Infants 0- 12 Weeks of Age

(a) Correlation with age (weeks)

No of samples Ratio r

Cord blood 50 $fa +0.2685 y/(Y -0.082 flly + 0.049

Infants 15 + 0.544

weeks Ply + 0.8637 0-12 7fcV - 0.8064

(b) Correlation with weight (grams)

P I P 0.05 < O . l + 0.0495 N.S. NS +0.052 NS NS + 0.2886 0.05 < 0.05 + 0.6428 0.001 < 0.05 < O . O O l - 0.6932 0.001 <0.05 < O . O O l + 0.8331 Ko.001

were obtained between the Ply ratios and the weight of newborn infants (r = t0.28, P < 0.05), Table I. When babies aged 0-12 weeks were examined, all three ratios showed significant correlation with age (weeks after birth ) or weight (grams) (Table I). Equal quantities of p and y chain occurred 5.2-6.8 weeks after birth.

DISCUSSION

HbF is the major hemoglobin in utero reaching up to 95% of the total hemoglobin during the second trimester and beginning to fall from the 34th week of pregnancy. In cord blood about 70-90% of total hemoglobin is HbF [ 2 , 3 ] . After birth it falls to adult levels of less than 1% by about 12 months of age. Various techniques are available for estimating HbF levels, including the alkali denaturation technique [4] as well as by enumeration of HbF containing cells by elution techniques [5] or immunological techniques [ 6 , 7 ] . The tech- nique of quantitation of globin chains described here provides another method for the esti- mation of various globin chains. In this study we have applied this technique to estimate the various globin chain ratios at various times in premature babies, cord blood, and in new- born infants. The technique has the advantage of allowing visual demonstration of abnormal hemoglobins during the quantitation procedure (hemoglobins like Hb S, C , etc, can be quan- titated in one procedure).

Although there is considerable scatter of various levels at various gestation ages, it can be concluded that a fall in the proportion of y/cr chain occurs at about 2,000 gm weight, and that about equal quantities of P and y chains are present about 5-6 weeks after birth. These figures agree with the findings for HbF as shown by Huehns and Beaven [ 3 ] , who find that 50% reduction of HbF occurs at about 2 months of age. I t has been shown that the fall of HbF is related to duration of gestation and the period of postnatal existence rather than time of birth, and hence that the disappearance of HbF is slower in premature infants, while in postmature infants there is a distinctly lower level of HbF than in normal [8-101 In our study there was significant correlation between Ply ratio and birth weight (P < 0.05). There was also significant correlation between the various ratios and either age or weight during the first 12 weeks after birth. It is important to note that the ratios reported here reflect overall production of a, f l , and y chains, not globin chain synthesis. Studies such as those re- ported by Bard [ 101 would still be required for the evaluation of globin synthesis at the time of study for eg, assessment of thalassemia status.

Quantitative Globin Electrophoresis 34 5

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7. Wood WG, Stamatoyannopoulos G , Lim G, Nute PE: F-cells in the adult: Normal values and levels in individuals with hereditary and acquired elevations of HbF. Blood, 46, 671-683, 1975.

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