Acta Pzdiutr Scund 64: 404408, 1975
STUDIES ON MATURITY IN NEWBORN INFANTS VII. Foetal Huemoglobin ORVAR FINNSTROM. L E I F G O T H E F O R S and BO Z E T T E R L U N D From the Drpurtments of Partiiatrics, University Hospituls of Linkoping und Umeri, Snzeden
ABSTRACT. Finnstrom, O., Gothefors, L. and Zetterlund, B. (Departments of Paediatrics, University Hospitals, Linkoping and Umel, Sweden). Studies on maturity in newborn infants. VII. Foetal haemoglohin. Acta Paediatr Scand, 64:404, 1 9 7 5 . 4 o r d blood from 125 newborns of various gestational ages has been analysed for the ratio foetal haemoglobin to total haemoglobin, using a n alkali denaturation method. The quotient, percentage of foetal haemoglobin divided by birth weight correlates well with gestational age. Thus the percentage of foetal haemoglohin in cord blood can be used as a method for estimating maturity in newborn infants. Foetal haemoglobin has been compared with other methods for maturity assessments and Seems to give the same precision in estimating gestational age as the best of these, which is the scoring of external characteristics. However, the latter method is considerably less timeconsuming and more suitable for routine use.
KEY WORDS: Maturity, gestational age, newborn infants, foetal haemoglobin
We have previously in a series of papers discussed the difficulty of estimating maturity (or gestational age) in newborn infants. The methods we used were anthropometric measDejinitions and abbreviations used Gestational age: age in days from the first day of the mother's last menstrual period until the day of birth. 40th week: days 274 to 280. SGA =small-for-gestational age. Infant with birth weight below-2 S.D. in t h e relation between birth weight and gestational age, according to Swedish standard curves (24). AGA=appropriate-for-gestational-age. Infant with birth weight within normal limitsfor the gestational age (between -2 and +2 S.D.). LGA=large-for-gestational age. Infant with birth weight above + 2 S.D. according to Swedish standard curves. Pre-term: gestational age less than 267 days postmenstrual. Term: gestational age between 267 and 294 days. Post-term: gestational age more than 294 days postmenstrual. Maturity: as used by the authors, maturity is an expression of the degree of development of the newborn infant. Maturity, thus defined, is dependent on the gestational age of the infant, but also on other factors such a s biological variation. Acta Psediatr Scand 64
urements-external characteristics, neurological examination, X-ray of epiphyseal centres and motor conduction velocity (6, 13, 14, 15, 16). Although planned, we were not able to include foetal haemoglobin (HbF) in these studies. In 1955, Cottom ( 1 1 ) seems to have been the first to describe a correlation between gestational 'age and the percentage of foetal haemoglobin in cord blood. In 1958, Brody (7) described foetal haemoglobin as a method of estimating maturity of newborn infants, and further investigated the method in 1960 (8). He was rather optimistic as to the value of this method. Later, several other authors ( 1 , 2, 5, 12, 19, 20, 21, 22, 25) have used foetal haemoglobin in the same situations as Brody but, as a rule, with less success. We therefore thought it might be of value t o perform a new study of this method, comparing foetal haemoglobin with some of the methods we used earlier, We also think that such comparison is
Maturity in newborn infants. VII
Table 1 . Important abnormal signs noticed in the invesduring the perjnatal tigated material Number Low foetal heart rate during delivery Postnatal asphyxia ADgar score below 7 at I minute Ap& score below 7 at 10 minutes IRDS Neurological symptoms Congenital heart defect Isoimmunisation (mostly anti-D) Hyperbi lirubinaemia (bilirubin above 15 mg/lOO ml)
5
6 I 5 3 I 10
19
essential, as it will otherwise be difficult to evaluate the method (17). MATERIALS Criteria f o r selection Only infants for whom reliable information about gestational age was available were included in the study. The criteria have been detailed earlier (13). Investigated material The material was collected during the period January 1972 to April 1973atthe University HospitalofUmeiasapartof a joint obstetric and paediatric study dealing with various aspects of foetal and newborn maturity. The material, divided into gestational age groups of 2 week intervals, is shown in Table 2. There were only 2 SGA infants, 116 infants were AGA and 2 were LGA. 5 infants could not be classified according to Swedish standard curves which do not considergestationalages below 33 weeks (225 days). 14 infants had slight to moderate dysmaturity signs (Clifford syndrome). No mother had had any serious disease related to the pregnancy. 7 mothers were diabetics and were treated with insulin throughout pregnancy. 6 ( 1 twin mother) had had moderate toxemia (diastolic blood pressure above 90 mmHg in combination with albiminuria). 10 mothers had had minor bleeding at some time during pregnancy. The frequencies of low foetal heart rate and low Apgar scores are seen in Table 1 as are also the most important abnormal signs during the neonatal period.
METHODS Birth weight, was recorded a s earlier described (13). Eight external characteristics were examined and scored on the first or second day of life by one of us (B. Z . ) using the technique earlier described (14). The interscoring reliability between two examiners was studied on 20 infants. The interscoring reliability was good, the difference amounting to a maximum of 2 points in 3 infants, 1 point in 8 infants and no difference in the remaining 9 infants.
405
Neuroli,Ricalexaminuti~~n o n d s o r i n g of 10 neurological signs was performed by one of us (0.F.) using the technique earlier described (15). Most infants were examined on day 5 , a few on day 4 or 5 , respectively. Foetal haemoglobin. A heparinized blood sample was collected from the umbilical cord immediately after clamping, and stored in a refrigerator until analysed. The analysis was performed on day I or 2 in 109 cases, on day 3 in 8 cases, on day 4 in 5 cases and on day 7 in 3 cases. Foetal haemoglobin was measured and calculated according to Brody's modification (7) of Betke's method (4) with some further modification as follows. 0. I ml of blood was first haemolysed in 200 ml ofdistilled water which contained O.W% NH,OH. The rate of alkali denaturation was determined in a Guilford spectrophotometer, type 300-N at 415 nm. 6.0 ml of blood hemolysate was added to the cell and the extension read (Eo).The sample was continuously kept in water bath at 37°C. Next 0.2 ml of 2 N NaOH was quickly pipetted into the cell, the content was mixed, and the extinction value (E,)read at 30 sec intervals for 10 min. The end point of denaturation was measured after about 1 hr. The percentage of normal haemoglobin at time t(Hb,) was calculated according to the formula given by Brody (8).
and correction was made for dilution by the alkali solution. The relative amounts of foetal hemoglobin were assessed according to Betke (4) by plotting the logarithms of the Hb,-values against time and extrapolating the linear part of the curve to zero time. Control of methods. Five samples of cord blood were stored for 1 week in a refrigerator at approximately +4"C. All these samples were analysed for foetal haemoglobin after I , 3 and 7 days storage. No significant changes in the relative amounts of foetal haemoglobin were noted during this time. It was thus considered of minor importance on which day during the neonatal week the samples were analysed, provided they had been stored in a refrigerator. It was noted, however, that the sample was more difficult to handle when stored more than 3 days, due to a great increase in viscosity. As a comparison, it can be mentioned that if samples were stored at -20°C there was a definite decrease in the relative amount of foetal haemoglobin even after 1 days storage. Ten samples were analysed according to Brody and also with our modified method, meaning that the samples were kept in a water bath at 37°C during the alkali denaturation, thus diminishing the time to complete denaturation from about 3 hours to I hour. No differences in the results were noted with these two methods.
RESULTS The mean values and standard deviations for percentage foetal haemoglobin of total haemoglobin are shown in Table 2, the infants being divided into gestational groups of 2 week Acra Pediatr Scand 64
406
0. Finnstriim et al.
Table 2. Mean and S.D. f o r percentage foetal haemoglobin. Infants divided into gestational age groups of 2 k>eekintervals
Mean S.D.
280 N=33
All infants N=125
89.76 I .72
89.79 2.88
86.90 2.48
85.46 3.56
78. I9 5.57
79.23 6.49
82.45 6.49
intervals. In Table 3, the mean values and for the 19 infants with hyperbilirubinaemia standard deviations for the quotient percentage (bilirubin above 15 mg/100 ml), and the 10 inof foetal haemoglobin divided by birth weight fants with iso-immunization. There were four (as suggested by Brody, 7, 8 ) are shown, the twin pairs in the study, three non-identical infants being divided in gestational age groups with only minor differences in their respecof 2 week intervals. tive H b F value, and one non-identical twin The individual values for percentage foetal pair with identical HbF values. haemoglobin, as well as the individual quotients plotted against gestational age, show that DISCUSSION the resulting correlations fit reasonably well into a linear one within the age limits studied. Discussion of the method Thus, the linear regression line for the correla- Our slight modification of Brody’s (7) method tion between the two values for foetal for foetal haemoglobin estimation does not afhaemoglobin and gestational age-as well as fect the results. It requires less time but is still for the other parameters studied and gesta- time-consuming. The storage of the blood in a tional age-were calculated. These correlation refrigerator for some days after birth also does coefficients are given in Table 4. not affect the results. Freezing the sample, on The best correlation coefficient was, for the the other hand, rapidly lowers the relative correlation between gestational age and exter- amount of foetal haemoglobin. Thus a cord nal score, slightly better than that for the corre- blood sample taken at delivery can be used later lation between the quotient percentage foetal in the neonatal week for maturity estimations, haemoglobinlbirth weight and gestational age. provided the sample has been stored in a reThe linear regression equation for Y (gesta- frigerator and has not been frozen. tional age) on X (percentage of foetal Discussion of results haemoglobin divided by birth weight) was: The available literature (1, 2, 3 , 5 , 7 , 8, 12, 19, Y=3 15- 1.74 S 21, 22, 25) provides conflicting opinions as The HbF values for the 14 infants with regards the value of determining foetal dysmaturity signs were evenly distributed haemoglobin in cord blood in order to estimate about the regression line; the same was true maturity in newborn infants. These diverging Table 3. Mean and S.D f o r percentage foetal haemoglobin divided by birth weight. Infants divided into gestational age groups o f 2 week intervals
Mean S.D. Actu Prediutr Scand 64
280 N=33
All infants N=125
54.94 9.25
43.86 7.71
35.09
28.82 4.82
23.37 3.46
21.59 2.98
28.29 9.81
8.11
Maturity in new.+orn infants. VZI
407
Table 4. Coefficients for the correlation betnieen gestational age and the parameters f o r maturity estimations under study
centres (17) it was shown that external characteristics and neurological tests gave the best precision in estimating gestational age. The precision was 53 weeks, The present reBirth weight 0.81 sults indicate that the HbF quotient gave about Faetal haernoglobin -0.62 Foetal haemoglobin/birth weight -0.85 the same precision in estimating gestational age External characteristics 0.87 as did external characteristics. Neurological tests 0.78 Measuring foetal haemoglobin is more time-consuming, however, than the scoring of external characteristics. To use columnar opinions mainly relate to different ways of expressing the value of foetal haemoglobin. chromatography would be still more tedious Brody showed (7, 8 ) that the quotient, than the alkali denaturation method. Thus, for percentage of foetal haemoglobin divided by practical purposes, the simple method based on birth weight, was considerably better corre- external characteristics seems to be superior to lated to gestational age than was the percentage the other methods so far described for maturity foetal haemoglobin alone, as did Gupta et al., estimations. In this series, neurological tests later (20).Most other authors did not perform correlated slightly less well to gestational age this division by birth weight. Brody also than either external characteristics or the HbF showed that this H b F quotient correlated much quotient. The correlation between birth weight better to gestational age than did birth weight, and gestational age was surprisingly good and again confirmed by Gupta et al. (70). of the same order as that for neurological tests. Kirschbaum, on the other hand, estimating However, there were very few infants with foetal haernoglobin by columnar chromato- retarded intra-uterine growth in this material, graphy, did not find this increase in as compared with the earlier one studied by the precision by dividing with birth weight ( 2 1 ). author. Because few infants with intra-uterine Bard et al. ( 2 ) compared estimation of foetal haemoglobin by columnar chromatography growth retardation were studied, noconclusion with the estimation of foetal haemoglobin can be drawn regarding the influence on the synthesis as determined by incubation with C 14 percentage of foetal haemoglobin of this comleucine. H e found a better precision in estimat- plication. Bard et al. ( 2 ) however, showed that ing gestational age using the second method. the proportion of foetal haemoglobin being He did not, however, calculate the H b F quo- synthesized in SGA infants, was greater than expected forgestational age. Thus it is possible tient. It thus seems essential t o divide the per- that the estimation of foetal haemoglobin in centage of foetal haemoglobin by the birth SGA infants can lead to an underestimation of weight if this estimation is to be used for as- gestational age. Salzberger et al. (231, however, sessing maturity in newborn infants. Most au- found identical values in dizygotic twins, in thors did not make any comparison with the spite of quite varying birth weight within some estimation of gestational age from birth weight pairs. Bromberg (9) noticed an increased or other methods for assessing maturity. W e percentage of foetal haemoglobin in cord blood have earlier pointed out the importance of in a group with chronic maternal hypoxia, making comparison with other methods in or- compared with'a control group. The gestational der to evaluate a given method for maturity as- ages of these infants were not given and thus sessment. In an earlier study comparing the groups are difficult t o compare. Garby et al. anthropometric measurements, external char- (18) found increased synthesis of foetal acteristics, neurological tests, motor con- haemoglobin in one SGA infant and in an infant duction velocity and X-ray of epiphyseal to a toxemic mother. In this study, hawever, Arta Prediatr Srand 64
408
0.Finnstrom et al.
we did not find any changes in expected H b F values in infants of mothers with toxemia or in infants with dysmaturity signs (Clifford syndrome). Further, Cook et at. (10) did not find any increase of HbF in infants with the Clifford syndrome, i.e. in probable placental insufficiency. Rhesus immunization does not affect the proportion of foetal haemoglobin synthesis ( 2 ) . In this study as well as most other studies, cord blood has been used for the estimation of foetal haemoglobin. It should be possible to use this method for maturity estimation even if cord blood is not available since Bard e t al. 1973 (3) showed that the birth process did not alter the rate of transition from H b F to HbA.
10.
II.
12. 13.
14.
15. 16.
17.
ACKNOWLEDGEMENTS This work was supported by a grant from “Prenatalforskningsfonden”. HbF analyses were performed by Mrs Yvonne Anderson.
REFERENCES I . Andrews, B. F. & Willet, G. P.: Fetal hemoglobin concentration in the newborn. Index of maturity and as supportive evidence for maternal fetal transfusion. Am J Ohstet Gynecol, 91: 85, 1965. 2. Bard, H., Makowski, E. L . , Meschia, G. & Battaglia, F. C.: The relative rates of synthesis of hemoglobins A and F in immature red cells of newborn infants. Pediatrics, 45: 766, 1970. 3. Bard, H.: Postnatal fetal and adult hemoglobin synthesis in early preterm newborn infants. J Clin Invest, 52: 1789, 1973. 4. Betke, K.: Der menschliche rote Blutfarhsioff. Springer, Berlin, Gottingen, Heidelberg 1954. 5. Bhoyroo, S. K., D’Souza, S. W . , Gordon, H . & Scopes, J. W.: Fetal haemoglobin in cord blood as estimated with two different techniques. J Obstet Gynaecol Br Commonws, 77: 238, 1970. 6. Blom, S. & Finnstrom, 0.: Studies on maturity in newborn infants. V. Motor conduction velocity. Neuropaediatrie. 3: 129, 1971. 7. Brody, S.: The intra-uterine age of the foetus at birth. Actu Obstet Gynecol Scand, 37: 374, 1958. 8. - Further studies on the reliability of a new method for the determination of the duration of pregnancy. J Obstet Gynecol Br Emp, 67: 819, I%O. 9. Bromberg, Y. M., Abrahamov, A. & Salzberger, M.: The effect of maternal anoxaemia on the foetal
Acta Pzdiatr Scand 64
18.
19.
20.
21.
22.
23.
24.
25.
haemoglobin of the newborn. J Ohstet Gynaecol Br Emp, 63: 875, 1956. Cook, C. D., Brodie, H . R. & Allen, D. W.: Measurement of fetal hemoglobin in newborn infants. Correlation with gestational age and intrauterine hypoxia. Pediatrics, 20: 272, 1957. Cottom. D. G.: Foetal haemoglobin and postmaturity. J Obstet Gynaecol Br Emp, 62: 945, 1955. Erkrath, F.-A,: Fetales Hamoglobin und Reifegrad des Neugeborenen. Zentralbl Gynaekol, 89: 401, 1967. Finnstrom, 0.:Studies on maturity in newborn infants. I . Birth weight, crown-heel length, head circumference and skull diameters in relation to gestational age. Acta Paediatr Scand, 60: 685, 1971. - Studies on maturity in newborn infants. 11. External characteristics.Acta Paediatr Scand, 61: 24, 1972. - Studies on maturity in newborn infants. 111. Neurological examination. Neuropaediatrie, 3:72, 1971. - Studies on maturity in newborn infants. IV. Postnatal radiological examination of epiphyseal centers. Neuropaediatrie, 3: 119, 1971. - Studies on maturity in newborn infants. VI. Comparison between different methods for maturity estimation. Acra Paediatr Scand, 61: 33, 1972. Garby, L., Sjolin. S. & Vuille, J.-C.: Studies on erythro-kinetics in infancy. 11. The relative rate of synthesis of haemoglobin F and haemoglobin A during the first months of life. Acta Paediatr Scand, 51: 245, 1962. Gerbie, A. B., DeCosta, E. J . & Reis, R. A.: Fetal hemoglobin as an index of maturity. Am J Obstet Gynecol, 78: 57, 1959. Gupta, H . L., Singh, H . , Dhatt. P. S., Mehta, H . C. & Verma, K. C.: Relationship of cord blood foetal hemoglobin to birth weight and length of gestation. Indian J Med Res, 61:903, 1973. Kirschbaum, T. H.: Fetal hemoglobin content of cord blood determined by column chromatography. Am J Obstet Gynecol, 84: 1375, 1962. Randhava, Z. I . , Hashmi, J. A. & Rahman, M. A.: Fetal hemoglobin levels in newborn infants. Journalof Pakistan Medical Association, 20: 258, 1970. Salzberger, M., Abrahamov, A. & Bromberg, Y . M.: Fetal and adult hemoglobin fractions in dizygotic twins. Exp Med Surg, 14: 130, 1956. Sterky, G.: Swedish standard curves for intrauterine growth. Pediatrics, 46: 7, 1970. Tuttle, A. H., de Lamerens, S. A. & Lane, D. M.: Studies on fetal hemoglobin synthesis in the newborn: Its application in determining gestational age. Pediatr Res, 2: 312, 1968.
Submitted Aug. 5 , 1974 Accepted Sept. 23, 1974
(0.F.) Dept. of Paediatrics University Hospital S-581 85 Linkoping Sweden
STUDIES ON MATURITY IN NEWBORN INFANTS VII. Foetal Huemoglobin ORVAR FINNSTROM. L E I F G O T H E F O R S and BO Z E T T E R L U N D From the Drpurtments of Partiiatrics, University Hospituls of Linkoping und Umeri, Snzeden
ABSTRACT. Finnstrom, O., Gothefors, L. and Zetterlund, B. (Departments of Paediatrics, University Hospitals, Linkoping and Umel, Sweden). Studies on maturity in newborn infants. VII. Foetal haemoglohin. Acta Paediatr Scand, 64:404, 1 9 7 5 . 4 o r d blood from 125 newborns of various gestational ages has been analysed for the ratio foetal haemoglobin to total haemoglobin, using a n alkali denaturation method. The quotient, percentage of foetal haemoglobin divided by birth weight correlates well with gestational age. Thus the percentage of foetal haemoglohin in cord blood can be used as a method for estimating maturity in newborn infants. Foetal haemoglobin has been compared with other methods for maturity assessments and Seems to give the same precision in estimating gestational age as the best of these, which is the scoring of external characteristics. However, the latter method is considerably less timeconsuming and more suitable for routine use.
KEY WORDS: Maturity, gestational age, newborn infants, foetal haemoglobin
We have previously in a series of papers discussed the difficulty of estimating maturity (or gestational age) in newborn infants. The methods we used were anthropometric measDejinitions and abbreviations used Gestational age: age in days from the first day of the mother's last menstrual period until the day of birth. 40th week: days 274 to 280. SGA =small-for-gestational age. Infant with birth weight below-2 S.D. in t h e relation between birth weight and gestational age, according to Swedish standard curves (24). AGA=appropriate-for-gestational-age. Infant with birth weight within normal limitsfor the gestational age (between -2 and +2 S.D.). LGA=large-for-gestational age. Infant with birth weight above + 2 S.D. according to Swedish standard curves. Pre-term: gestational age less than 267 days postmenstrual. Term: gestational age between 267 and 294 days. Post-term: gestational age more than 294 days postmenstrual. Maturity: as used by the authors, maturity is an expression of the degree of development of the newborn infant. Maturity, thus defined, is dependent on the gestational age of the infant, but also on other factors such a s biological variation. Acta Psediatr Scand 64
urements-external characteristics, neurological examination, X-ray of epiphyseal centres and motor conduction velocity (6, 13, 14, 15, 16). Although planned, we were not able to include foetal haemoglobin (HbF) in these studies. In 1955, Cottom ( 1 1 ) seems to have been the first to describe a correlation between gestational 'age and the percentage of foetal haemoglobin in cord blood. In 1958, Brody (7) described foetal haemoglobin as a method of estimating maturity of newborn infants, and further investigated the method in 1960 (8). He was rather optimistic as to the value of this method. Later, several other authors ( 1 , 2, 5, 12, 19, 20, 21, 22, 25) have used foetal haemoglobin in the same situations as Brody but, as a rule, with less success. We therefore thought it might be of value t o perform a new study of this method, comparing foetal haemoglobin with some of the methods we used earlier, We also think that such comparison is
Maturity in newborn infants. VII
Table 1 . Important abnormal signs noticed in the invesduring the perjnatal tigated material Number Low foetal heart rate during delivery Postnatal asphyxia ADgar score below 7 at I minute Ap& score below 7 at 10 minutes IRDS Neurological symptoms Congenital heart defect Isoimmunisation (mostly anti-D) Hyperbi lirubinaemia (bilirubin above 15 mg/lOO ml)
5
6 I 5 3 I 10
19
essential, as it will otherwise be difficult to evaluate the method (17). MATERIALS Criteria f o r selection Only infants for whom reliable information about gestational age was available were included in the study. The criteria have been detailed earlier (13). Investigated material The material was collected during the period January 1972 to April 1973atthe University HospitalofUmeiasapartof a joint obstetric and paediatric study dealing with various aspects of foetal and newborn maturity. The material, divided into gestational age groups of 2 week intervals, is shown in Table 2. There were only 2 SGA infants, 116 infants were AGA and 2 were LGA. 5 infants could not be classified according to Swedish standard curves which do not considergestationalages below 33 weeks (225 days). 14 infants had slight to moderate dysmaturity signs (Clifford syndrome). No mother had had any serious disease related to the pregnancy. 7 mothers were diabetics and were treated with insulin throughout pregnancy. 6 ( 1 twin mother) had had moderate toxemia (diastolic blood pressure above 90 mmHg in combination with albiminuria). 10 mothers had had minor bleeding at some time during pregnancy. The frequencies of low foetal heart rate and low Apgar scores are seen in Table 1 as are also the most important abnormal signs during the neonatal period.
METHODS Birth weight, was recorded a s earlier described (13). Eight external characteristics were examined and scored on the first or second day of life by one of us (B. Z . ) using the technique earlier described (14). The interscoring reliability between two examiners was studied on 20 infants. The interscoring reliability was good, the difference amounting to a maximum of 2 points in 3 infants, 1 point in 8 infants and no difference in the remaining 9 infants.
405
Neuroli,Ricalexaminuti~~n o n d s o r i n g of 10 neurological signs was performed by one of us (0.F.) using the technique earlier described (15). Most infants were examined on day 5 , a few on day 4 or 5 , respectively. Foetal haemoglobin. A heparinized blood sample was collected from the umbilical cord immediately after clamping, and stored in a refrigerator until analysed. The analysis was performed on day I or 2 in 109 cases, on day 3 in 8 cases, on day 4 in 5 cases and on day 7 in 3 cases. Foetal haemoglobin was measured and calculated according to Brody's modification (7) of Betke's method (4) with some further modification as follows. 0. I ml of blood was first haemolysed in 200 ml ofdistilled water which contained O.W% NH,OH. The rate of alkali denaturation was determined in a Guilford spectrophotometer, type 300-N at 415 nm. 6.0 ml of blood hemolysate was added to the cell and the extension read (Eo).The sample was continuously kept in water bath at 37°C. Next 0.2 ml of 2 N NaOH was quickly pipetted into the cell, the content was mixed, and the extinction value (E,)read at 30 sec intervals for 10 min. The end point of denaturation was measured after about 1 hr. The percentage of normal haemoglobin at time t(Hb,) was calculated according to the formula given by Brody (8).
and correction was made for dilution by the alkali solution. The relative amounts of foetal hemoglobin were assessed according to Betke (4) by plotting the logarithms of the Hb,-values against time and extrapolating the linear part of the curve to zero time. Control of methods. Five samples of cord blood were stored for 1 week in a refrigerator at approximately +4"C. All these samples were analysed for foetal haemoglobin after I , 3 and 7 days storage. No significant changes in the relative amounts of foetal haemoglobin were noted during this time. It was thus considered of minor importance on which day during the neonatal week the samples were analysed, provided they had been stored in a refrigerator. It was noted, however, that the sample was more difficult to handle when stored more than 3 days, due to a great increase in viscosity. As a comparison, it can be mentioned that if samples were stored at -20°C there was a definite decrease in the relative amount of foetal haemoglobin even after 1 days storage. Ten samples were analysed according to Brody and also with our modified method, meaning that the samples were kept in a water bath at 37°C during the alkali denaturation, thus diminishing the time to complete denaturation from about 3 hours to I hour. No differences in the results were noted with these two methods.
RESULTS The mean values and standard deviations for percentage foetal haemoglobin of total haemoglobin are shown in Table 2, the infants being divided into gestational groups of 2 week Acra Pediatr Scand 64
406
0. Finnstriim et al.
Table 2. Mean and S.D. f o r percentage foetal haemoglobin. Infants divided into gestational age groups of 2 k>eekintervals
Mean S.D.
280 N=33
All infants N=125
89.76 I .72
89.79 2.88
86.90 2.48
85.46 3.56
78. I9 5.57
79.23 6.49
82.45 6.49
intervals. In Table 3, the mean values and for the 19 infants with hyperbilirubinaemia standard deviations for the quotient percentage (bilirubin above 15 mg/100 ml), and the 10 inof foetal haemoglobin divided by birth weight fants with iso-immunization. There were four (as suggested by Brody, 7, 8 ) are shown, the twin pairs in the study, three non-identical infants being divided in gestational age groups with only minor differences in their respecof 2 week intervals. tive H b F value, and one non-identical twin The individual values for percentage foetal pair with identical HbF values. haemoglobin, as well as the individual quotients plotted against gestational age, show that DISCUSSION the resulting correlations fit reasonably well into a linear one within the age limits studied. Discussion of the method Thus, the linear regression line for the correla- Our slight modification of Brody’s (7) method tion between the two values for foetal for foetal haemoglobin estimation does not afhaemoglobin and gestational age-as well as fect the results. It requires less time but is still for the other parameters studied and gesta- time-consuming. The storage of the blood in a tional age-were calculated. These correlation refrigerator for some days after birth also does coefficients are given in Table 4. not affect the results. Freezing the sample, on The best correlation coefficient was, for the the other hand, rapidly lowers the relative correlation between gestational age and exter- amount of foetal haemoglobin. Thus a cord nal score, slightly better than that for the corre- blood sample taken at delivery can be used later lation between the quotient percentage foetal in the neonatal week for maturity estimations, haemoglobinlbirth weight and gestational age. provided the sample has been stored in a reThe linear regression equation for Y (gesta- frigerator and has not been frozen. tional age) on X (percentage of foetal Discussion of results haemoglobin divided by birth weight) was: The available literature (1, 2, 3 , 5 , 7 , 8, 12, 19, Y=3 15- 1.74 S 21, 22, 25) provides conflicting opinions as The HbF values for the 14 infants with regards the value of determining foetal dysmaturity signs were evenly distributed haemoglobin in cord blood in order to estimate about the regression line; the same was true maturity in newborn infants. These diverging Table 3. Mean and S.D f o r percentage foetal haemoglobin divided by birth weight. Infants divided into gestational age groups o f 2 week intervals
Mean S.D. Actu Prediutr Scand 64
280 N=33
All infants N=125
54.94 9.25
43.86 7.71
35.09
28.82 4.82
23.37 3.46
21.59 2.98
28.29 9.81
8.11
Maturity in new.+orn infants. VZI
407
Table 4. Coefficients for the correlation betnieen gestational age and the parameters f o r maturity estimations under study
centres (17) it was shown that external characteristics and neurological tests gave the best precision in estimating gestational age. The precision was 53 weeks, The present reBirth weight 0.81 sults indicate that the HbF quotient gave about Faetal haernoglobin -0.62 Foetal haemoglobin/birth weight -0.85 the same precision in estimating gestational age External characteristics 0.87 as did external characteristics. Neurological tests 0.78 Measuring foetal haemoglobin is more time-consuming, however, than the scoring of external characteristics. To use columnar opinions mainly relate to different ways of expressing the value of foetal haemoglobin. chromatography would be still more tedious Brody showed (7, 8 ) that the quotient, than the alkali denaturation method. Thus, for percentage of foetal haemoglobin divided by practical purposes, the simple method based on birth weight, was considerably better corre- external characteristics seems to be superior to lated to gestational age than was the percentage the other methods so far described for maturity foetal haemoglobin alone, as did Gupta et al., estimations. In this series, neurological tests later (20).Most other authors did not perform correlated slightly less well to gestational age this division by birth weight. Brody also than either external characteristics or the HbF showed that this H b F quotient correlated much quotient. The correlation between birth weight better to gestational age than did birth weight, and gestational age was surprisingly good and again confirmed by Gupta et al. (70). of the same order as that for neurological tests. Kirschbaum, on the other hand, estimating However, there were very few infants with foetal haernoglobin by columnar chromato- retarded intra-uterine growth in this material, graphy, did not find this increase in as compared with the earlier one studied by the precision by dividing with birth weight ( 2 1 ). author. Because few infants with intra-uterine Bard et al. ( 2 ) compared estimation of foetal haemoglobin by columnar chromatography growth retardation were studied, noconclusion with the estimation of foetal haemoglobin can be drawn regarding the influence on the synthesis as determined by incubation with C 14 percentage of foetal haemoglobin of this comleucine. H e found a better precision in estimat- plication. Bard et al. ( 2 ) however, showed that ing gestational age using the second method. the proportion of foetal haemoglobin being He did not, however, calculate the H b F quo- synthesized in SGA infants, was greater than expected forgestational age. Thus it is possible tient. It thus seems essential t o divide the per- that the estimation of foetal haemoglobin in centage of foetal haemoglobin by the birth SGA infants can lead to an underestimation of weight if this estimation is to be used for as- gestational age. Salzberger et al. (231, however, sessing maturity in newborn infants. Most au- found identical values in dizygotic twins, in thors did not make any comparison with the spite of quite varying birth weight within some estimation of gestational age from birth weight pairs. Bromberg (9) noticed an increased or other methods for assessing maturity. W e percentage of foetal haemoglobin in cord blood have earlier pointed out the importance of in a group with chronic maternal hypoxia, making comparison with other methods in or- compared with'a control group. The gestational der to evaluate a given method for maturity as- ages of these infants were not given and thus sessment. In an earlier study comparing the groups are difficult t o compare. Garby et al. anthropometric measurements, external char- (18) found increased synthesis of foetal acteristics, neurological tests, motor con- haemoglobin in one SGA infant and in an infant duction velocity and X-ray of epiphyseal to a toxemic mother. In this study, hawever, Arta Prediatr Srand 64
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we did not find any changes in expected H b F values in infants of mothers with toxemia or in infants with dysmaturity signs (Clifford syndrome). Further, Cook et at. (10) did not find any increase of HbF in infants with the Clifford syndrome, i.e. in probable placental insufficiency. Rhesus immunization does not affect the proportion of foetal haemoglobin synthesis ( 2 ) . In this study as well as most other studies, cord blood has been used for the estimation of foetal haemoglobin. It should be possible to use this method for maturity estimation even if cord blood is not available since Bard e t al. 1973 (3) showed that the birth process did not alter the rate of transition from H b F to HbA.
10.
II.
12. 13.
14.
15. 16.
17.
ACKNOWLEDGEMENTS This work was supported by a grant from “Prenatalforskningsfonden”. HbF analyses were performed by Mrs Yvonne Anderson.
REFERENCES I . Andrews, B. F. & Willet, G. P.: Fetal hemoglobin concentration in the newborn. Index of maturity and as supportive evidence for maternal fetal transfusion. Am J Ohstet Gynecol, 91: 85, 1965. 2. Bard, H., Makowski, E. L . , Meschia, G. & Battaglia, F. C.: The relative rates of synthesis of hemoglobins A and F in immature red cells of newborn infants. Pediatrics, 45: 766, 1970. 3. Bard, H.: Postnatal fetal and adult hemoglobin synthesis in early preterm newborn infants. J Clin Invest, 52: 1789, 1973. 4. Betke, K.: Der menschliche rote Blutfarhsioff. Springer, Berlin, Gottingen, Heidelberg 1954. 5. Bhoyroo, S. K., D’Souza, S. W . , Gordon, H . & Scopes, J. W.: Fetal haemoglobin in cord blood as estimated with two different techniques. J Obstet Gynaecol Br Commonws, 77: 238, 1970. 6. Blom, S. & Finnstrom, 0.: Studies on maturity in newborn infants. V. Motor conduction velocity. Neuropaediatrie. 3: 129, 1971. 7. Brody, S.: The intra-uterine age of the foetus at birth. Actu Obstet Gynecol Scand, 37: 374, 1958. 8. - Further studies on the reliability of a new method for the determination of the duration of pregnancy. J Obstet Gynecol Br Emp, 67: 819, I%O. 9. Bromberg, Y. M., Abrahamov, A. & Salzberger, M.: The effect of maternal anoxaemia on the foetal
Acta Pzdiatr Scand 64
18.
19.
20.
21.
22.
23.
24.
25.
haemoglobin of the newborn. J Ohstet Gynaecol Br Emp, 63: 875, 1956. Cook, C. D., Brodie, H . R. & Allen, D. W.: Measurement of fetal hemoglobin in newborn infants. Correlation with gestational age and intrauterine hypoxia. Pediatrics, 20: 272, 1957. Cottom. D. G.: Foetal haemoglobin and postmaturity. J Obstet Gynaecol Br Emp, 62: 945, 1955. Erkrath, F.-A,: Fetales Hamoglobin und Reifegrad des Neugeborenen. Zentralbl Gynaekol, 89: 401, 1967. Finnstrom, 0.:Studies on maturity in newborn infants. I . Birth weight, crown-heel length, head circumference and skull diameters in relation to gestational age. Acta Paediatr Scand, 60: 685, 1971. - Studies on maturity in newborn infants. 11. External characteristics.Acta Paediatr Scand, 61: 24, 1972. - Studies on maturity in newborn infants. 111. Neurological examination. Neuropaediatrie, 3:72, 1971. - Studies on maturity in newborn infants. IV. Postnatal radiological examination of epiphyseal centers. Neuropaediatrie, 3: 119, 1971. - Studies on maturity in newborn infants. VI. Comparison between different methods for maturity estimation. Acra Paediatr Scand, 61: 33, 1972. Garby, L., Sjolin. S. & Vuille, J.-C.: Studies on erythro-kinetics in infancy. 11. The relative rate of synthesis of haemoglobin F and haemoglobin A during the first months of life. Acta Paediatr Scand, 51: 245, 1962. Gerbie, A. B., DeCosta, E. J . & Reis, R. A.: Fetal hemoglobin as an index of maturity. Am J Obstet Gynecol, 78: 57, 1959. Gupta, H . L., Singh, H . , Dhatt. P. S., Mehta, H . C. & Verma, K. C.: Relationship of cord blood foetal hemoglobin to birth weight and length of gestation. Indian J Med Res, 61:903, 1973. Kirschbaum, T. H.: Fetal hemoglobin content of cord blood determined by column chromatography. Am J Obstet Gynecol, 84: 1375, 1962. Randhava, Z. I . , Hashmi, J. A. & Rahman, M. A.: Fetal hemoglobin levels in newborn infants. Journalof Pakistan Medical Association, 20: 258, 1970. Salzberger, M., Abrahamov, A. & Bromberg, Y . M.: Fetal and adult hemoglobin fractions in dizygotic twins. Exp Med Surg, 14: 130, 1956. Sterky, G.: Swedish standard curves for intrauterine growth. Pediatrics, 46: 7, 1970. Tuttle, A. H., de Lamerens, S. A. & Lane, D. M.: Studies on fetal hemoglobin synthesis in the newborn: Its application in determining gestational age. Pediatr Res, 2: 312, 1968.
Submitted Aug. 5 , 1974 Accepted Sept. 23, 1974
(0.F.) Dept. of Paediatrics University Hospital S-581 85 Linkoping Sweden
