Studies of fetal hemogolobin have been undertaken during the past one hundred years and have been reported by a number of investigators.
Von Korber (1866) found the blood from the umbilical cord of the placenta had more resistance against alkaline reagents than did adult blood. This has been repeatedly confirmed by von Kruger (1888), Wakulenko (1910) and Helpern (1943) etc.
In 1933, Eastman et al and Leibson et al (1936) stated that, in comparison with adult blood fetal blood more strongly combined with oxygen and more readily released the carbon fetal blood more strongly combined with oxygen and more readily released the carbon dioxide. In recent years it has become clear that fetal
hemoglobin has a different hemical structure from adult hemoglobin. they can be spearated by electrophoresis, by chromatography and by immunological procedures. Also Chernoff and Singer (1952) using an alkali denaturation method, observed the decrease in the amount of fetal hemoglobin as the age increase from the new born to the adult. Kleihauser et al (1957) made clear that, adult hemoglobin could be removed from the erythrocytes of venous blood by use of citric acid-phosphate
buffer. Then only fetal hemoglobin remained and could be separately stained and observed. Employing this technic, in normal children and adults, Zipursky et al (1962) observed the changes in the percentage of erythrocytes containing fetal hemoglobin
However, the studies on fetal hemoglobin in the Korean population have been limited so far to: (1) Dr.Pyung Il Kwon (1962) who measured the amount of fetal hemoglobin in the blood of umbilical cord, and correlated the results with the duration of pregnancy and body weight etc. (2) Dr. Chang Eui Hong and Hae Won Lee (1963) who measured the amount of fetal hemoglobin in different age groups of normal Koreans, and (3) Dr. Woon Kee Lee (1934) who identified fetal erythrocytes in the maternal blood.
In this study, using different age groups of Korean children and adults, the author observed changes in the amount of fetal hemoglobin in venous blood and changes in the number of fetal erythrocytes. Also these two series of studies were correlated.
Materials and Methods
During the 22 months from October 1963 to July 1865, venous blood specimens were collected from 214 normal healthty Korean children and adults who were examined at the Yonsei University of Medicine Severance Hospital, E-Hwa University Medical College Hospital, Cheil Hospital. Seoul City Children's Hospital and the World of Vision Children's Center.
For each specimen, approximately 4 ml. of venous blood was collected into a bottle containing 6 mg. of potassium oxalate to prevent coagulation. The following procedures were carried out.
(1) For each case fetal hemoglobin was determined by the Singer and Cheroff's alkali denaturation method; ie. 3 ml. of blood was centrifuged to remove the plasam. After washing the packed cells three times with 0.85% saline, then 1.4ml. of distilled water and 0.4ml. of toluene were added to each ml. of packed red blood cells. This mixture was shaken vigorously for five minutes and then centrifuged. The upper toluene layer was gently removed by aspiration and the lower hemoglobin layer was filtered through Whatman No. 44 filter paper. For each sample, 0.02ml. of filtrate was diluted by adding 5 ml. of distilled water. Optical density (ie. O.D. of total hemoglo bin) of this solution was read using a spectrophotometer at the wave length of 540 millimicron. Then, at a 20℃. temperature, 0.1ml. of hemoglobin solution (the filtrate) was added to 1.6ml. of 1/12 N.KOH solution. Exactly one minute after this, 3.4ml. of half saturated ammonium sulfate solution was added and filtered through Whatman No.44 filter paper. The optical density of the filtrate (ie. the O.D. of fetal hemoglobin) was read at 540 millimicron. A calculation of
the percentage of the amount of fetal hemoglobin was done by the following equation: Fetal hemoglobin (%) = O.D. of fetal hemoglobin / O.D. of total hemoglobin x 20.3
(2) From the 214 cases studied above, 99 cases under 5 year of age and 21 cases selected at random from those over 5 years after birth (a total 120 cases) were examined for the changes in the amount of fetal hemoglobin in the erythrocytes using the Betke and Kleihauser's method (1958): ie. 0.1ml. of blood was diluted 1:3 with physiological saline and smeared on slide glasses. After drying, they were fixed for 2 minutes with ethyl alcohol (95% or more) and washed for 90 seconds in a citric acid-dibasic potassium phosphate buffer solution (pH. 3.48-33.52). The
smears were then dried and stained with Wright's stain. A microscopic examination was done on these smears. Those erythrocytes containing adult hemoglobin revealed only an outline of the cells with a white center. Those erythrocytes containing
fetal hemoglobin were red. The depth of redness varied according to the amounts of fetal hemoglobin contained in the individual erythrocytes. Those erythrocytes containing adult hemoglobin were scaled as grade O, While those erythrocytes containing fetal hemoglobin were graded 1,2,3, or 4 according to the depth of
staining. 200 to 500 cells were counted for each case when the fetal erythrocytes were more than 1%. 1,000 to 20,000 cells were examined when the fetal red blood cells found were less than 1%. The percentage of fetal erythrocytes found were calculated.
The average values of the fetal hemoglobin obtained by the alkali denaturation test were: 71.6% for the fetal bloods, 64.8% for the first month after birth, 20.4% for 1-3 months, 5.5% for 3-6 months, 4.5% for 6-12 months, 2.7% for 1-2 years, 2.1% for 2-3 years, 1.5% for 3-4 years and 1.1 to 1.2% for the groups between 4 to 50 years or more.
The average values of fetal erythrocytes obtained by the Betke and Kleihauser's method were: 96.3% for the fetal blood, 93.8% for the first month, 81.4% for the second month, 78% for the third month, 27.6% for the 3-6 months, 4.7% for the 6-9 months, 1.07% for the 9-12 months, 0.2% for the 1-2 years, 0.05% for 2-3 years, 0.013% for 3-4 years and 0.008% for the 4-5 years.
The group which was old 5 years or more had two cases, each of which contained one fetal erythrocyte after a search of approximately 20,000 erythrocytes in each case.
Discussion and summary
The aforementioned results obtained in this study can be summarized as follows:
1) The average amounts of fetal hemoglobin of normal Koreans was 71.6% at birth, but as age increased the amount diminished rater rapidly to reach 5.5% during the period of 3-6 months after birth, 1.5% during 3-4 years, and aveeerage values of 1.1% to 1.2% were maintained after four years of age.
2) The average values of fetal erythrocytes of normal Koreans were 96.3% at birth and as the age increased the value diminished gradually to reach an average value of 27.6% ddduring 3-6 months, 0.2% during 1-2 years and 0.008% during 4-5 years after birth, and for the group over four years of age only two of the 21 cases
revealed a single fetal erythrocyte.
In comparing the above two series it is concluded that, at the time of birth the fetal hemoglobin and adult hemoglobin are present simultaneously in the same erythrocyte (at least in some of the cells). As the age increases both the amount of fetal hemoglobin and the percentage of fetal erythrocytes diminish, but in all cases over one year of age studied in this series, the amount (%) of fetal hemoglobin exceeded the number (%) of fetal erythrocytes found.
This fact indicates that adult erythrocytes, at least in some of the cells, contain fetal hemoglobin in addition to the adult hemoglobin, and the variation in staining intensity of fetal cells indicate that individual erythrocytes contain various proportions of fetal hemoglobin and adult hemoglobin.
This means that normal erythrocyte precursors can possess the potentiality of forming both hemoglobin A and hemoglobin F.Before birth the formation of Hemoglobin F predominates while after birth the formation of Hemoglobin A predominates. It also indicates that the same erythrocytic precursors can form gamma polypeptide as well as beta polypeptide of the hemoglobin molecule.