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Fetal hemoglobin content of cord blood determined by column chromatography
Fetal hemoglobin content of cord blood determined by column chromatography THOMAS H. KIRSCHBAUM, M.D. Salt Lake City, Utah
T H E heterogeneity of human blood pigment has been known for nearly 100 years. 1 A technique for the objective measurement of the relative proportions of alkaline resistant and alkaline labile fractions has been available for nearly that long, 2 yet problems in the measurement of fetal hemoglobin and its appropriate evaluation continue. Though it is clear that the blood pigment of the immature human fetus is predominantly of fetal type, estimates of its quantity vary widely among investigators. A report of a trace of adult hemoglobin in a 2~ month premature fetus, 3 the demonstration of 17 and 27 per cent adult hemoglobin m 2 ten-week human fetuses, and a range of from 7 to 4 7 per cent adult hemoglobin in 15 fetuses less than 20 weeks' gestation 4 • 5 should be contrasted with the statement of Jonxis 6 that adult hemoglobin is present only in minor quantities in infants weighing less than 1,000 grams at birth. Though the percentage content of fetal hemoglobin of fetal blood decreases progressively through the third trimester of pregnancy, the standard deviation from the mean at any given period of gestation ranges from 2 to 20 per cent of the mean value with techniques reported to date. 7- 13 In addition, the figures cited for the mean percentage fetal hemoglobin content of cord blood obtained at
term from normal infants range from 50 to 98 per cent. 6 • 7 • 10 • 13 - 18 Though fetal hemoglobin is produced in normal infants postnatally and in augmented quantities in some instances of disease/· 14• "1 9 - 22 this variability would not seem to explain the estimates of postnatal age at which fetal hemoglobin becomes no longer demonstrable since this value is variously reported as 22 weeks, 18 28 tn
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and 5 to 6 years. Though early immunologic techniques failed to demonstrate any fetal hemoglobin in the blood of normal adults, 3 more recent applications of this method have revealed measurable quantities. 20 Alkaline denaturation techniques are compatible with quantities of fetal hemoglobin in adult blood which are too small to measure with real precision. 6 • 20 - 25 The differences in properties of adult and fetal hemoglobins have been extensively summarized in recent years. 6 • 1·8 • 21 • 26 • 27 Despite real differences between these two forms of hemoglobin with respect to electrophoretic behavior, paper chromatographic mobility, solubi!ity of carbonmonoxyhemoglobin and ferrihemoglobin, ultraviolet spectrophotometry, amino acid sequence, immunologic specificity, isoleucine content, crsytallization form, and the rate of spreading of monomolecular films, few of these have served as useful bases for clinical determinations. This stems either from the relatively minute difference between the two forms on analylsis, from the complexity of the equipment necessary, or the difficulties in control and performance
From the Department of Obstetrics and Gynecology, University of Utah Medical School. This work is supported by Research Grant No. H-4962 from the National Institutes of Health.
1375
1376 Kirschbaum
of techniques. Almost invariably then. the resistance of fetal hemoglobin to denaturation on exposure to dilute solutions of alkali has been used in the determination of percentage content of fetal hemoglobin for clinical purposes. The most commonly employed techniques are those of Brinkman and Jonxis 23 and the later modification of Jonxis and Visser,Z 4 the method of Singer, Chernoff, and Singer/ 2 and that of Betke. 28 Investigative work in this country has employed predominantly the method of Singer, Chernoff, and Singer, or that of Brinkman and Jonxis and it is clear that, with either of these techniques, low concentrations of fetal hemoglobin, in the range of 5 to 10 per cent, produce results of questionable validity.U· 21 The comparative study of Jonxis and Huisman29 has demonstrated consistent errors in both procedures. The Singer method produces values which are too low when the percentage of fetal hemoglobin is more than 50, and too high for lower values. The method of Brinkman and Jonxis produces erratic values at the low range of percentage fetal hemoglobin. The reproducibility of duplicate determination with either of these methods would seem to approximate plus or minus 5 per cent. 8 • 21 A measure of the usefulness of fetal hemoglobin determinations comes from evaluation of the relationship between percentage fetal hemoglobin content of fetal blood and the duration of gestation. The standard deviation for values of percentage fetal hemoglobin between the thirty-sixth and fortyfourth weeks of gestation would seem to be approximately plus or minus 10 per cent of the mean figure. 13 · 16 • 30 The reproducibility of values obtained by the method of Betke appears to be significantly better in the experience of Brody. 9 It is clear that the use of this method in determination of percentage fetal hemoglobin as a function of the duration of gestation yields results which are not useful for the prediction of gestational age from the related variable. 11 - 13 Because of this, an attempt was made to evaluate a protocol for the determination of fetal hemoglobin employing the differences
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in behavior of fetal and adult hemoglobin in column chromatographv using IRC 50 a.~ described by Boardman and Partridgen in bovine and caprinc species and used extensively for separation and purification of human adult hemoglobin by Morrison and Cook, 32 Huisman and Prins, 33 Prins and Huisman, 34 Schroeder, 3 " Prins/ 6 and Hunt. 37 Experimental method
Amberlite IRC 50 synthetic cation exchange resin, Lot No. 792246, was prepared according to the method of Hirs, Moore, and Stein. 38 This involves wet sizing the resin to collect approximately 1 Kg. of the particle size passed by a size 200 mesh but retained by a size 400 mesh. This material is then repeatedly washed with 4 to 5 washes of 2 L. of water per kilogram resin, the supernatant decanted, and the resin air dried. It is then treated with a similar volume of acetone, filtered, and subjected to multiple acetone washings and once again air dried. The material is next suspended in water and treated with 40 per cent sodium hydroxide with the cessation of exothermia as the end point. The alkaline form is then washed repeatedly with water until the filtrate approaches pH 10, and converted to the acidic form with 10 L. of 3N hydrochloric acid. The material is next washed with water repeatedly and brought to a pH approximating buffer pH by sodium hydroxide back titration. Multiple washes with buffer then follow until the pH stability of the effluent demonstrates equilibrium between buffer and resin. A phosphate-potassium cyanide buffer was employed having a final pH of 7.02 at 25° C. sodium concentration of 0.05M and potassium cyanide concentration O.OlM. The composition is the same as that described by Allen, Schroeder, and Balog35 as buffer No.3 (NaH 2P0 4 ·H 2 0 3.45 Gm., Na 2HP0 4 1.78 Gm. KCN 0.65 Gm. in 1 L. H 2 0). This buffer pH appears to be optimal in terms of producing maximal separation between adult and fetal fractions over the relatively short column length employed. A column of 4 em. length glass tubing with O.D. 8 mm. I.D. 6 mm. leading to a seg-
Volume 84 Number 10
ment of capillary tubing with I.D. 1.5 mm. was used. The base of the column was supported by a small plug of glass wool. Cord blood was obtained at the time of birth in a series of 172 deliveries. Blood was withdrawn into a heparinized syringe and washed 4 times with normal saline. The erythrocyte residue was mixed with an excess volume of distilled water and thorough mixing was obtained. The specimen was then centrifuged and the supernatant filtered once through Schleicker and Schnelll'"~o. 597 filter paper. All procedures thereafter were carried out at 5° C. The hemoglobin solution was dialyzed in Visking tubing against buffer for a period of at least 24 hours, the resultant solution being diluted as appropriate with cold buffer to a concentration range between 2.5 and 3.5 mg. per 0.2 ml. The approximation of Winegarden and Borsook to compute concentration from optical absorption at 541 mp. was used. 39 Repeat determinations had demonstrated this to be the optimal quantity of hemoglobin added in 0.2 ml. volume to the column to allow clear separation of the two hemoglobin fractions, yet not overload the column nor produce effluent concentrations too small to be measured spectrophotometrically. A Beckman Model DU spectrophotometer with a hydrogen light source was employed for measurements. The absorption spectra, both of adult and fetal blood fractions, were determined and the presence of a sharp peak at 415 mp. confirmed on this instrument for both adult and fetal ferrihemoglobin cyanide samples. This wave length was used for measurements, and 4 ml. fractions of column effluent were read in a quartz cuvette. Fraction separation appeared to be roughly independent of flow rate and a rate of effluent flow between 7 and 15 ml. per hour was employed throughout. Under these conditions, with chromatography being performed at 5° C., an initial hemoglobin fraction begins to appear in effluent volumes past 1 ml. and the entirety of this fraction is contained within the first 20 ml. of effluent buffer, at least 90 per cent being contained in the initial 5 mi. In view
Fetal hemoglobin content of coid blood
1377
of its alkaline resistance, this will be designated as the fetal hemoglobin fraction. Adult hemoglobin fractions begin to emerge from the column somewhere after 30 to 40 ml. of effluent volume has passed and hemoglobin is eluted sufficiently slowly so that continued collection was not feasible for the entirety of the fraction. If after collection of the fetal hemoglobin fraction, the column, supernatant buffer, and absorbed hemoglobin are allowed to equilibrate vvith room temperature so that the effluent temperature approaches 25° C., the entire residual hemoglobin is then eluted within the period of time necessary for passage of 5 ml. of effluent volume. In this fashion, at 5° C., the IRC 50 column may be used as a trap for adult hemoglobin allowing the free passage of fetal hemoglobin within the time limits designated. The column is then interrupted by clamping an attached segment of rubber tubing. The system is allowed to return to room temperature with little fear of the production of methemoglobin in view of the tenacity of formation of the ferrihemoglobin cyanide complex, and the hemoglobin remaining in the column eluted rapidly. The measurement of effluent volumes and the estimation of optical density at 415 mp. of these two fractions allows the caculation of the relative proportion of fetal hemoglobin with respect to total hemoglobin in the initial sample_ Serial 5 mi. volumes were collected from the column under the above conditions and each of them subjected to analysis using the Jonxis and Visser modification of the Brinkman-Jonxis method at a pH of 12.7, reading the optical density at 576 mp.. 24 Seven cord blood samples were chromatographed, with assay by alkaline denaturation of portions of resulting fractions. In each of 7 sets of determinations, the initial fractions described as fetal hemoglobin fractions above, behaved uniformly as fetal hemoglobin with a constant rate of denaturation and the position of the denaturation curve corresponding to 100 per cent fetal hemoglobin on extrapolation. Though the slope of the curve varied in minor fashion from one sample to
1378
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Fig. 1. Per cent fetal hemoglobin content of cord blood as a function of the duration of gestation.
50
200
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240
260
280
320
OAYS GESTATION
Fig. 2. Per cent fetal hemoglobin of cord blood per unit birth weight as a function of the duration of gestation.
another, this could be demonstrated to be related to the concentration of hemoglobin contained. On the other hand, the late fractions obtained uniformly demonstrated no undenatured hemoglobin 1 minute after exposure to sodium hydroxide of appropriate concentration. This was compatible with the proposition that fetal hemoglobin content in the so-called adult fractions was less than 5 per cent. In a series of 42 duplicate determinations of percentage fetal hemoglobin, the mean value of duplicates was determined and the percentage deviation of each value from this mean was obtained. The range for the deviations from the mean was 0 per cent to 1.61 per cent of the mean and the mean deviation was 0.47 per cent, denoting a strong corespondence of duplicate values. Fetal hemoglobin determinations were correlated with the duration of gestation obtained from the menstrual history of the patient, evaluation of fetal weight, length, and clinical evidence of maturity. Where data was questionable in any regard, the case was discarded. This al1owed the consideration of 153 samples of accurate gestational age.
Mean and standard deviations of percentagP fetal hemoglobin as a function of the weeks of gestation arc shown in Table I. A plot of percentage fetal hemoglobin as a function of days' gestation is shown in Fig. 1. Treating that portion of the curve which corresponds to gestation greater than 250 days, the coefficient of linear correlation is -0.625. With a t value of 7.44, this yields a probability of random occurrence less than 1 per million. The equation of the regression line of y on x is y = 170.30- 0.31x, where y is percentage fetal hemoglobin and x is days' gestation. The 95 per cent confidence range for values of x at the mean value of y, that is 83.9 per cent, is plus or minus 13.9 days. Because of the suggestion of Brody 9 · 11 that the ratio of percentage fetal hemoglobin to body weight is a more reliable correlate of duration of gestation, a plot of these 2 variables was obtained and is shown in Fig. 2. For duration of gestation greater than 250 days, the linear coefficient of correlation is -0.615 and corresponds to a probability of random occurrence of less than 1 in 1 million. The regression equation for this portion of the distribution is y = 98.56 - eJ.26x, where y is percentage fetal hemoglobin divided by birth weight in kilograms, and x is days' gestation. The mean value of the ordinate 26.1 corresponds to a zone of 95 per cent confidence which encompasses plus or minus 18 days. Eighteen samples of normal adult human blood were analyzed, and yielded a mean percentage of fast-running hemoglobin of 10.2 per cent, with standard deviation of 3.2 per cent. Three cases of active erythroblastosis fetalis, as defined by jaundice and anemia in the newborn, direct Coombs'-positive Rhpositive fetal erythrocytes, and maternal blood Rh negative \vith positive indirect Coombs test using Rh-positive test cells, were studied. These produced, at 280, 289, and 298 days' gestation, values of percentage fetal hemoglobin of 86.4 per cent, 79.1 per cent, and 74.6 per cent, respectively.
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Comment
The composition of early- and late-running fractions of hemoglobin in the system as described is known to be complex. The so-called fetal fraction includes an extremely rapidly running component which is a nonheme protein and represents roughly 2 per cent of the total protein chormatographed. 35 Because this fraction absorbs maximally at 280 mp. and relatively little at 415 mJ.t and because it represents a relatively small proportion of the total protein, ·it produces no significant error in the data. The relative proportion of the nonheme protein fraction may be influenced by deviation from reduced temperature conditions which would augment denaturation of hemoglobin without its full incorporation into the ferrihemoglobin cyanide complex. The major portion of fetal hemoglobin, defined in terms of resistance to alkaline denaturation, occurs in two fractions in this system/5 the first, F 1 , comprising approximately 20 per cent of the total fetal hemoglobin, the second, F 2 , representing the remainder. The possibility of heterogeneity of fetal hemoglobin as a function of the duration of gestation has been suggested by Kiinzer 40 and similar findings have been reported by Halbrecht, Klibanski, Brzoza, and Lahov,4 • 5 using paper electrophoresis, as well as Halbrecht, Klibanski, and Bar Ilan using column chromatographyY A discussion of these findings and a description of the fractions of fetal hemoglobin demonstrated in a column chromatography system similar to the one in this report has recently been published by Matsuda, Schroeder, Jones, and Weliky. 42 Heterogeneity of fetal hemoglobin is similarly implied by Brinkman and Jonxis23 and White, Delory, and Israels 43 on the basis of alkaline denaturation techniques. Evidence for the heterogeneity of normal adult human hemoglobin is also clear and stems from the initial observation on starch block electrophoresis by Kunkel and Wallenius,44 though the same phenomenon was described by Brinkman who used alkaline denaturation techniques at pH of 11:7 ear-
Fetol hemoglobin content of cord blood 1379
lier. 18 Itano 45 has recently reviewed this subject in detail. Chromatographic techniques readily demonstrate this finding as the results on the IRC 50 column using a phosphate-citrate buffer at pH 6.3 in the work of Morrison and Cook 32 and a similar finding by Prins36 would show. Similar data have been published by Huisman, Martis, and Dozy/6 as well as by Gutter, Peterson, and Sober, 47 using a carboxymethylcellulose column. The most detailed work in this area appears to be that by Allen, Schroeder, and Balog 35 and by Clegg and Schroeder/ 8 in which normal human adult hemoglobin has been separated into 7 fractions using an IRC 50 column and a phosphate cyanide buffer with pH 6.85. The A 1 fraction which in turn is composed of 5 subfractions represents approximately 10 to 15 per cent of the protein content of a chromatographed sample and lies within range of the so-called fetal hemoglobin fraction in this modified system. This observation is confirmed in the present study at pH 7.02 by the analysis of samples of normal human adult blood. The resulting mean percentage of fast-running hemoglobin of 10.2 per cent compares well with the results of others. 32 • 35 • 36 • 48 The presence of this material serves as an effective bar to the use of this technique in the evaluation of adult blood and represents a predictable error in the determination applied to fetal blood such that the per-
Table I. Mean values of percentage fetal hemoglobin of cord blood in relation to weeks' gestation computed from menstrual data Weeks' f(estation
Less than 36 ( 182 to 246 days) 36 37 38
39 40 41 42 43
I
No. of Mean %--fetal cases , hemoglobin
7 3 7 18 28 39 25 17 9
Standard de viations
92.3
0.9
89.6 90.1 88.6 85.6 84.2 81.9 78.8 77.4
1.6 3.3 3.0 4.6 4.3 5.4 3.7 3.1
1380 Kirschbaum
centage fetal hemoglobin is falsely high by a factor which is roughly 10 per cent of the adult hemoglobin content. Despite the probable presence of adult hemoglobin in the fast-running fractions collected at 5° C. with buffer pH 7.02, the method has some real advantages at its present preliminary state in terms of the evaluation of cord blood. The reproducibility of duplicate determinations is 5 to 10 times better than that which has been previously reported with the use of alkaline denaturation techniques. Any estimate of the duration of gestation has inherent in it predictable errors. These stem from such independent variables as the duration of time between menses and subsequent ovulation, the discrepancy in date of last menstrual period arising unintentionally from memory defect or from patient deception, and the intrinsic variation of the duration of pregnancy. As Schild bach suggests, 49 from calculations based on Stewart's data, 50 the basic variation in duration of gestation using basal body temperature change to denote the time of ovulation preceding conception is such as to produce a 95 per cent confidence range of approximately plus or minus 14.3 days. In the present study, the 95 per cent confidence range taken at the mean value of percentage fetal hemoglobin for the sample is plus or minus 13.9 days. In contrast, similar data derived from a plot of percentage fetal hemoglobin determined by the alkaline denaturation technique of Betke, and fetal age, in the hands of Brody, yields a range of 95 per cent confidence of plus or minus 53.1 days. The confidence range is decreased to plus or minus 18.5 days in Brody's study by using as the independent variable, the percentage fetal hemoglobin divided by fetal weight at the time of
."\ovemhcr 15, 1962 Am. J. Obst. & Gynec.
birth. It is clear that the modified column chromatographic technique produces a similar range of accuracy with unmodified measurements and that this range of accuracy approaches the probable theoretical limit of estimate of the duration of gestation Fig. 1 demonstrates a linear decrease in the percentage content of fetal hemoglobin of cord blood past a period of 260 days' gestation through 307 days' gestation. Though there are relatively few measurements prior to that time, they are compatible with the hypothesis that percentage fetal hemoglobin content is constant, at least past 182 days of gestation, and approximates 95 per cent of total hemoglobin. Though again the data are insufficient to demonstrate this without question, it is entirely possible that some decline begins prior to the two hundred fiftieth day of gestation. The apparent depression of the percentage content of fetal hemoglobin in the cord blood of babies involved in active erythroblastosis fetalis has been demonstrated many times since the report of Schulman and Smith. 51 Though only 3 cases of active erythroblastosis fetalis were studied in the present series, the percentage content of fetal hemoglobin from these bloods in no significant way deviates from the norm for the appropriate period of gestation. Though no significance can be attached to an observation based on 3 cases, it would suggest the need to repeat the fetal hemoglobin assay of cord blood in this disease by some method other than alkaline denaturation. The method at present is being reported in terms of preliminary findings. It might be considered to show promise in terms of the high order of reproducibility of repeat determinations and the low order of scatter as a function of the duration of gestation.
REFERENCES
1. von Korber, E.: Inaugural Dissertation, Darpat, 1866, cited by Bischoff, H.: Ztschr. ges. exper. Med. 48: 472, 1926. 2. von Kriiger, F.: Ztschr. Bioi. 24: 318, 1887. 3. Darrow, R. R., Nowakovsky, S., and Austin, M. H.: Arch. Path. 30: 873, 1940.
4. Halbrecht, 1., and Klibanski, Ch.: Nature, London 178: 794, 1956. 5. Halbrecht, I., Klibanski, Ch., Brzoza, H., and Lahov, M.: Am. ]. Clin. Path. 29: 340, 1958. 6. Jonxis, J. H. P.: Abnormal Hemoglobins,
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7.
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23. 24. 25. 26. 27. 28.
Springfield, Ill., 1959, Charles C Thomas, Publisher, p. 114. Beaven, G. H., Hoch, H., and Holiday, ·E. R.: Biochem. J. 49: 374, 1951. Cottom, D. G.: J. Obst. & Gynaec. Brit. Emp. 62: 945, 1955. Brody, 8.: Acta obst. et gynec. scandinav. 37: 374, 1958. Gerbie, A. B., DeCosta, E. J., and Reis, R. A.: AM. J. 0BST. & GYNEC. 78: 57, 1959. Brody, S.: J. Obst. & Gynaec. Brit. Emp. 67: 819, 1960. Brody, 8.: J. Obst. & Gynaec. Brit. Emp. 67: 672, 1960. Agiiero, 0.: Obst. & Gynec. 19: 257, 1962. Chernoff, A. I., and Singer, K.: Pediatrics 9: 469, 1952. Schulman, I., and Smith, C. A.: A. M. A. Am. J. Dis. Child. 86: 354, 1953. Bromberg, Y. M., Abrahamov, A., and Salzberger, M.: J. Obst. & Gynaec. Brit. Emp. 63: 875, 1956. Abrahamov, A., Salzberger, M., and Bromberg, Y. M.: Am. J. Clin. Path. 26: 146, 1956. Jonxis, J. H. P.: In Roughton, F. J. W., and Kendrew, J. C., editors: Haemoglobin, London, 1949, Butterworth & Co., Ltd., p. 261. Baar, H. S., and Hickmans, E. M.: ]. Physiol. 100: 3P, 194 L Chernoff, A. I.: Blood 8:413, 1953. White, J. C., and Beaven, G. H.: J. Clin. Path. 7: 175, 1954. Singer, K., Chernoff, A. L and Singer, L.: Blood 6: 413, 1951. Brinkman, R., and Jonxis, J. H. P.: J. Physiol. 85: 117, 1935. Jonxis, J. H. P., and Visser, H. K. A.: A. M. A. J. Dis. Child. 92: 588, 1956. Robinson, A. R., Robson, M., Harrison, A. P., and Zuelzer, W. W.: J. Lab. & Clin. Med. 50: 745, 1957. Leeks, H., and Wolman, I. J.: Am. ]. M. Sc. 219: 684, 1950. Ingram, V. M.: Hemoglobin and Its Abnormalities, Springfield, Ill., 1961, Charles C Thomas, Publisher. Betke, K., and Sorelsberg, W.: Biochem. Ztschr. 320: 431, 1950.
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29. Jonxis, J. H. P., and Huisman, T. H. J.: Blood 11: 1009, 1956. 30. Walker, ].: Cold Spring Harbor Symposia of Quantitative Biology, XIX, Baltimore, Maryland, 1954, Monumental Printing Co., p. 141. 31. Boardman, U. K., and Partridge, S. M.: Biochem. ]. 59: 543, 1955. 32. Morrison, M., and Cook, J. L.: Science 122: 920, 1955. 33. Huisman, T. H. J., and Prins, H. K.: J. Lab. & Clin. Med. 46: 255, 1955. 34. Prins, H. K., and Huisman, T. H. J.: Nature, London 175: 903, 1955. 35. Allen, D. W., Schroeder, W. A., and Balog, J.: J. Am. Chem Soc. 80: 1628, 1958. 36. Prins, H. K.: Chromatografische Onderzoekingen over Menselijke Haemoglobinen, Groningen, 1958, Drukkerij I. Oppenheim, N. V. 37. Hunt, J. A.: Nature, London 183: 1373, 1959. 38. Hirs, C. H. W., Moore, S., and Stein, W. H.: J. Bioi. Chern. 200: 493, 1953. 39. Winegarden, H. W., and Borsook, H.: J. Cell. & Comp. Physiol. 3: 437, 1939. 40. Kiinzer, W.: Nature, London 179: 477, 1957. 41. Halbrecht, I., Klibanski, Ch., and Bar Ilan, F.: Nature, London 183: 327, 1959. 42. Matsuda, C., Schroeder. W. A., Jo!l.es, R. T., and Weliky, N.: Blood 16: 984, 1960. 43. White, F. D., Delory, G. E., and Israels, L. G.: Canad. J. Res. 28: 231, 1950. 44. Kunkel, H. G., and Wallenius, G.: Science 122: 288, 1955. 45. Itano, H.: Am. Rev. Biochem. 25: 331, 1956. 46. Huisman, T. H . .T., Martis, E. A., and Dozy, A.: J. Lab. & Clin. Med. 52: 312, 1958. 47. Gutter, F. J., Peterson. E. A., and Sober, H. A.: Arch. Biochem. 80: 353, 1959. 48. Clegg, M. D., and Schroeder, W. A.: ]. Am. Chern. Soc. 81: 6065, 1959. 49. Schildbach, H. R.: Klin. Wchnschr. 31: 654, 1953. 50. Stewart, H. L., Jr.: J. A. M. A. 148: 1079, 1952. 51. Schulman, I., and Smith, C. A.: A. M. A. Am. J. Dis. Child. 87: 167, 1954. University of Utah Co!lege of Medicine Salt Lake City 15, Utah
T H E heterogeneity of human blood pigment has been known for nearly 100 years. 1 A technique for the objective measurement of the relative proportions of alkaline resistant and alkaline labile fractions has been available for nearly that long, 2 yet problems in the measurement of fetal hemoglobin and its appropriate evaluation continue. Though it is clear that the blood pigment of the immature human fetus is predominantly of fetal type, estimates of its quantity vary widely among investigators. A report of a trace of adult hemoglobin in a 2~ month premature fetus, 3 the demonstration of 17 and 27 per cent adult hemoglobin m 2 ten-week human fetuses, and a range of from 7 to 4 7 per cent adult hemoglobin in 15 fetuses less than 20 weeks' gestation 4 • 5 should be contrasted with the statement of Jonxis 6 that adult hemoglobin is present only in minor quantities in infants weighing less than 1,000 grams at birth. Though the percentage content of fetal hemoglobin of fetal blood decreases progressively through the third trimester of pregnancy, the standard deviation from the mean at any given period of gestation ranges from 2 to 20 per cent of the mean value with techniques reported to date. 7- 13 In addition, the figures cited for the mean percentage fetal hemoglobin content of cord blood obtained at
term from normal infants range from 50 to 98 per cent. 6 • 7 • 10 • 13 - 18 Though fetal hemoglobin is produced in normal infants postnatally and in augmented quantities in some instances of disease/· 14• "1 9 - 22 this variability would not seem to explain the estimates of postnatal age at which fetal hemoglobin becomes no longer demonstrable since this value is variously reported as 22 weeks, 18 28 tn
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and 5 to 6 years. Though early immunologic techniques failed to demonstrate any fetal hemoglobin in the blood of normal adults, 3 more recent applications of this method have revealed measurable quantities. 20 Alkaline denaturation techniques are compatible with quantities of fetal hemoglobin in adult blood which are too small to measure with real precision. 6 • 20 - 25 The differences in properties of adult and fetal hemoglobins have been extensively summarized in recent years. 6 • 1·8 • 21 • 26 • 27 Despite real differences between these two forms of hemoglobin with respect to electrophoretic behavior, paper chromatographic mobility, solubi!ity of carbonmonoxyhemoglobin and ferrihemoglobin, ultraviolet spectrophotometry, amino acid sequence, immunologic specificity, isoleucine content, crsytallization form, and the rate of spreading of monomolecular films, few of these have served as useful bases for clinical determinations. This stems either from the relatively minute difference between the two forms on analylsis, from the complexity of the equipment necessary, or the difficulties in control and performance
From the Department of Obstetrics and Gynecology, University of Utah Medical School. This work is supported by Research Grant No. H-4962 from the National Institutes of Health.
1375
1376 Kirschbaum
of techniques. Almost invariably then. the resistance of fetal hemoglobin to denaturation on exposure to dilute solutions of alkali has been used in the determination of percentage content of fetal hemoglobin for clinical purposes. The most commonly employed techniques are those of Brinkman and Jonxis 23 and the later modification of Jonxis and Visser,Z 4 the method of Singer, Chernoff, and Singer/ 2 and that of Betke. 28 Investigative work in this country has employed predominantly the method of Singer, Chernoff, and Singer, or that of Brinkman and Jonxis and it is clear that, with either of these techniques, low concentrations of fetal hemoglobin, in the range of 5 to 10 per cent, produce results of questionable validity.U· 21 The comparative study of Jonxis and Huisman29 has demonstrated consistent errors in both procedures. The Singer method produces values which are too low when the percentage of fetal hemoglobin is more than 50, and too high for lower values. The method of Brinkman and Jonxis produces erratic values at the low range of percentage fetal hemoglobin. The reproducibility of duplicate determination with either of these methods would seem to approximate plus or minus 5 per cent. 8 • 21 A measure of the usefulness of fetal hemoglobin determinations comes from evaluation of the relationship between percentage fetal hemoglobin content of fetal blood and the duration of gestation. The standard deviation for values of percentage fetal hemoglobin between the thirty-sixth and fortyfourth weeks of gestation would seem to be approximately plus or minus 10 per cent of the mean figure. 13 · 16 • 30 The reproducibility of values obtained by the method of Betke appears to be significantly better in the experience of Brody. 9 It is clear that the use of this method in determination of percentage fetal hemoglobin as a function of the duration of gestation yields results which are not useful for the prediction of gestational age from the related variable. 11 - 13 Because of this, an attempt was made to evaluate a protocol for the determination of fetal hemoglobin employing the differences
11. 196~ Ob>t. ,\ C)nec.
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l
in behavior of fetal and adult hemoglobin in column chromatographv using IRC 50 a.~ described by Boardman and Partridgen in bovine and caprinc species and used extensively for separation and purification of human adult hemoglobin by Morrison and Cook, 32 Huisman and Prins, 33 Prins and Huisman, 34 Schroeder, 3 " Prins/ 6 and Hunt. 37 Experimental method
Amberlite IRC 50 synthetic cation exchange resin, Lot No. 792246, was prepared according to the method of Hirs, Moore, and Stein. 38 This involves wet sizing the resin to collect approximately 1 Kg. of the particle size passed by a size 200 mesh but retained by a size 400 mesh. This material is then repeatedly washed with 4 to 5 washes of 2 L. of water per kilogram resin, the supernatant decanted, and the resin air dried. It is then treated with a similar volume of acetone, filtered, and subjected to multiple acetone washings and once again air dried. The material is next suspended in water and treated with 40 per cent sodium hydroxide with the cessation of exothermia as the end point. The alkaline form is then washed repeatedly with water until the filtrate approaches pH 10, and converted to the acidic form with 10 L. of 3N hydrochloric acid. The material is next washed with water repeatedly and brought to a pH approximating buffer pH by sodium hydroxide back titration. Multiple washes with buffer then follow until the pH stability of the effluent demonstrates equilibrium between buffer and resin. A phosphate-potassium cyanide buffer was employed having a final pH of 7.02 at 25° C. sodium concentration of 0.05M and potassium cyanide concentration O.OlM. The composition is the same as that described by Allen, Schroeder, and Balog35 as buffer No.3 (NaH 2P0 4 ·H 2 0 3.45 Gm., Na 2HP0 4 1.78 Gm. KCN 0.65 Gm. in 1 L. H 2 0). This buffer pH appears to be optimal in terms of producing maximal separation between adult and fetal fractions over the relatively short column length employed. A column of 4 em. length glass tubing with O.D. 8 mm. I.D. 6 mm. leading to a seg-
Volume 84 Number 10
ment of capillary tubing with I.D. 1.5 mm. was used. The base of the column was supported by a small plug of glass wool. Cord blood was obtained at the time of birth in a series of 172 deliveries. Blood was withdrawn into a heparinized syringe and washed 4 times with normal saline. The erythrocyte residue was mixed with an excess volume of distilled water and thorough mixing was obtained. The specimen was then centrifuged and the supernatant filtered once through Schleicker and Schnelll'"~o. 597 filter paper. All procedures thereafter were carried out at 5° C. The hemoglobin solution was dialyzed in Visking tubing against buffer for a period of at least 24 hours, the resultant solution being diluted as appropriate with cold buffer to a concentration range between 2.5 and 3.5 mg. per 0.2 ml. The approximation of Winegarden and Borsook to compute concentration from optical absorption at 541 mp. was used. 39 Repeat determinations had demonstrated this to be the optimal quantity of hemoglobin added in 0.2 ml. volume to the column to allow clear separation of the two hemoglobin fractions, yet not overload the column nor produce effluent concentrations too small to be measured spectrophotometrically. A Beckman Model DU spectrophotometer with a hydrogen light source was employed for measurements. The absorption spectra, both of adult and fetal blood fractions, were determined and the presence of a sharp peak at 415 mp. confirmed on this instrument for both adult and fetal ferrihemoglobin cyanide samples. This wave length was used for measurements, and 4 ml. fractions of column effluent were read in a quartz cuvette. Fraction separation appeared to be roughly independent of flow rate and a rate of effluent flow between 7 and 15 ml. per hour was employed throughout. Under these conditions, with chromatography being performed at 5° C., an initial hemoglobin fraction begins to appear in effluent volumes past 1 ml. and the entirety of this fraction is contained within the first 20 ml. of effluent buffer, at least 90 per cent being contained in the initial 5 mi. In view
Fetal hemoglobin content of coid blood
1377
of its alkaline resistance, this will be designated as the fetal hemoglobin fraction. Adult hemoglobin fractions begin to emerge from the column somewhere after 30 to 40 ml. of effluent volume has passed and hemoglobin is eluted sufficiently slowly so that continued collection was not feasible for the entirety of the fraction. If after collection of the fetal hemoglobin fraction, the column, supernatant buffer, and absorbed hemoglobin are allowed to equilibrate vvith room temperature so that the effluent temperature approaches 25° C., the entire residual hemoglobin is then eluted within the period of time necessary for passage of 5 ml. of effluent volume. In this fashion, at 5° C., the IRC 50 column may be used as a trap for adult hemoglobin allowing the free passage of fetal hemoglobin within the time limits designated. The column is then interrupted by clamping an attached segment of rubber tubing. The system is allowed to return to room temperature with little fear of the production of methemoglobin in view of the tenacity of formation of the ferrihemoglobin cyanide complex, and the hemoglobin remaining in the column eluted rapidly. The measurement of effluent volumes and the estimation of optical density at 415 mp. of these two fractions allows the caculation of the relative proportion of fetal hemoglobin with respect to total hemoglobin in the initial sample_ Serial 5 mi. volumes were collected from the column under the above conditions and each of them subjected to analysis using the Jonxis and Visser modification of the Brinkman-Jonxis method at a pH of 12.7, reading the optical density at 576 mp.. 24 Seven cord blood samples were chromatographed, with assay by alkaline denaturation of portions of resulting fractions. In each of 7 sets of determinations, the initial fractions described as fetal hemoglobin fractions above, behaved uniformly as fetal hemoglobin with a constant rate of denaturation and the position of the denaturation curve corresponding to 100 per cent fetal hemoglobin on extrapolation. Though the slope of the curve varied in minor fashion from one sample to
1378
:\o\j'lllb~·~
v: •• ~hh~ .. ~
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.\m .
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Results
:~~<·.
90,-
;,. i i
~
"'60~
·-·~·. ·. ", ,.· . .
.
7J 160
200
220
240
260
260
300
320
DAYS GESTATION
Fig. 1. Per cent fetal hemoglobin content of cord blood as a function of the duration of gestation.
50
200
220
240
260
280
320
OAYS GESTATION
Fig. 2. Per cent fetal hemoglobin of cord blood per unit birth weight as a function of the duration of gestation.
another, this could be demonstrated to be related to the concentration of hemoglobin contained. On the other hand, the late fractions obtained uniformly demonstrated no undenatured hemoglobin 1 minute after exposure to sodium hydroxide of appropriate concentration. This was compatible with the proposition that fetal hemoglobin content in the so-called adult fractions was less than 5 per cent. In a series of 42 duplicate determinations of percentage fetal hemoglobin, the mean value of duplicates was determined and the percentage deviation of each value from this mean was obtained. The range for the deviations from the mean was 0 per cent to 1.61 per cent of the mean and the mean deviation was 0.47 per cent, denoting a strong corespondence of duplicate values. Fetal hemoglobin determinations were correlated with the duration of gestation obtained from the menstrual history of the patient, evaluation of fetal weight, length, and clinical evidence of maturity. Where data was questionable in any regard, the case was discarded. This al1owed the consideration of 153 samples of accurate gestational age.
Mean and standard deviations of percentagP fetal hemoglobin as a function of the weeks of gestation arc shown in Table I. A plot of percentage fetal hemoglobin as a function of days' gestation is shown in Fig. 1. Treating that portion of the curve which corresponds to gestation greater than 250 days, the coefficient of linear correlation is -0.625. With a t value of 7.44, this yields a probability of random occurrence less than 1 per million. The equation of the regression line of y on x is y = 170.30- 0.31x, where y is percentage fetal hemoglobin and x is days' gestation. The 95 per cent confidence range for values of x at the mean value of y, that is 83.9 per cent, is plus or minus 13.9 days. Because of the suggestion of Brody 9 · 11 that the ratio of percentage fetal hemoglobin to body weight is a more reliable correlate of duration of gestation, a plot of these 2 variables was obtained and is shown in Fig. 2. For duration of gestation greater than 250 days, the linear coefficient of correlation is -0.615 and corresponds to a probability of random occurrence of less than 1 in 1 million. The regression equation for this portion of the distribution is y = 98.56 - eJ.26x, where y is percentage fetal hemoglobin divided by birth weight in kilograms, and x is days' gestation. The mean value of the ordinate 26.1 corresponds to a zone of 95 per cent confidence which encompasses plus or minus 18 days. Eighteen samples of normal adult human blood were analyzed, and yielded a mean percentage of fast-running hemoglobin of 10.2 per cent, with standard deviation of 3.2 per cent. Three cases of active erythroblastosis fetalis, as defined by jaundice and anemia in the newborn, direct Coombs'-positive Rhpositive fetal erythrocytes, and maternal blood Rh negative \vith positive indirect Coombs test using Rh-positive test cells, were studied. These produced, at 280, 289, and 298 days' gestation, values of percentage fetal hemoglobin of 86.4 per cent, 79.1 per cent, and 74.6 per cent, respectively.
Volume 84 Number 10
Comment
The composition of early- and late-running fractions of hemoglobin in the system as described is known to be complex. The so-called fetal fraction includes an extremely rapidly running component which is a nonheme protein and represents roughly 2 per cent of the total protein chormatographed. 35 Because this fraction absorbs maximally at 280 mp. and relatively little at 415 mJ.t and because it represents a relatively small proportion of the total protein, ·it produces no significant error in the data. The relative proportion of the nonheme protein fraction may be influenced by deviation from reduced temperature conditions which would augment denaturation of hemoglobin without its full incorporation into the ferrihemoglobin cyanide complex. The major portion of fetal hemoglobin, defined in terms of resistance to alkaline denaturation, occurs in two fractions in this system/5 the first, F 1 , comprising approximately 20 per cent of the total fetal hemoglobin, the second, F 2 , representing the remainder. The possibility of heterogeneity of fetal hemoglobin as a function of the duration of gestation has been suggested by Kiinzer 40 and similar findings have been reported by Halbrecht, Klibanski, Brzoza, and Lahov,4 • 5 using paper electrophoresis, as well as Halbrecht, Klibanski, and Bar Ilan using column chromatographyY A discussion of these findings and a description of the fractions of fetal hemoglobin demonstrated in a column chromatography system similar to the one in this report has recently been published by Matsuda, Schroeder, Jones, and Weliky. 42 Heterogeneity of fetal hemoglobin is similarly implied by Brinkman and Jonxis23 and White, Delory, and Israels 43 on the basis of alkaline denaturation techniques. Evidence for the heterogeneity of normal adult human hemoglobin is also clear and stems from the initial observation on starch block electrophoresis by Kunkel and Wallenius,44 though the same phenomenon was described by Brinkman who used alkaline denaturation techniques at pH of 11:7 ear-
Fetol hemoglobin content of cord blood 1379
lier. 18 Itano 45 has recently reviewed this subject in detail. Chromatographic techniques readily demonstrate this finding as the results on the IRC 50 column using a phosphate-citrate buffer at pH 6.3 in the work of Morrison and Cook 32 and a similar finding by Prins36 would show. Similar data have been published by Huisman, Martis, and Dozy/6 as well as by Gutter, Peterson, and Sober, 47 using a carboxymethylcellulose column. The most detailed work in this area appears to be that by Allen, Schroeder, and Balog 35 and by Clegg and Schroeder/ 8 in which normal human adult hemoglobin has been separated into 7 fractions using an IRC 50 column and a phosphate cyanide buffer with pH 6.85. The A 1 fraction which in turn is composed of 5 subfractions represents approximately 10 to 15 per cent of the protein content of a chromatographed sample and lies within range of the so-called fetal hemoglobin fraction in this modified system. This observation is confirmed in the present study at pH 7.02 by the analysis of samples of normal human adult blood. The resulting mean percentage of fast-running hemoglobin of 10.2 per cent compares well with the results of others. 32 • 35 • 36 • 48 The presence of this material serves as an effective bar to the use of this technique in the evaluation of adult blood and represents a predictable error in the determination applied to fetal blood such that the per-
Table I. Mean values of percentage fetal hemoglobin of cord blood in relation to weeks' gestation computed from menstrual data Weeks' f(estation
Less than 36 ( 182 to 246 days) 36 37 38
39 40 41 42 43
I
No. of Mean %--fetal cases , hemoglobin
7 3 7 18 28 39 25 17 9
Standard de viations
92.3
0.9
89.6 90.1 88.6 85.6 84.2 81.9 78.8 77.4
1.6 3.3 3.0 4.6 4.3 5.4 3.7 3.1
1380 Kirschbaum
centage fetal hemoglobin is falsely high by a factor which is roughly 10 per cent of the adult hemoglobin content. Despite the probable presence of adult hemoglobin in the fast-running fractions collected at 5° C. with buffer pH 7.02, the method has some real advantages at its present preliminary state in terms of the evaluation of cord blood. The reproducibility of duplicate determinations is 5 to 10 times better than that which has been previously reported with the use of alkaline denaturation techniques. Any estimate of the duration of gestation has inherent in it predictable errors. These stem from such independent variables as the duration of time between menses and subsequent ovulation, the discrepancy in date of last menstrual period arising unintentionally from memory defect or from patient deception, and the intrinsic variation of the duration of pregnancy. As Schild bach suggests, 49 from calculations based on Stewart's data, 50 the basic variation in duration of gestation using basal body temperature change to denote the time of ovulation preceding conception is such as to produce a 95 per cent confidence range of approximately plus or minus 14.3 days. In the present study, the 95 per cent confidence range taken at the mean value of percentage fetal hemoglobin for the sample is plus or minus 13.9 days. In contrast, similar data derived from a plot of percentage fetal hemoglobin determined by the alkaline denaturation technique of Betke, and fetal age, in the hands of Brody, yields a range of 95 per cent confidence of plus or minus 53.1 days. The confidence range is decreased to plus or minus 18.5 days in Brody's study by using as the independent variable, the percentage fetal hemoglobin divided by fetal weight at the time of
."\ovemhcr 15, 1962 Am. J. Obst. & Gynec.
birth. It is clear that the modified column chromatographic technique produces a similar range of accuracy with unmodified measurements and that this range of accuracy approaches the probable theoretical limit of estimate of the duration of gestation Fig. 1 demonstrates a linear decrease in the percentage content of fetal hemoglobin of cord blood past a period of 260 days' gestation through 307 days' gestation. Though there are relatively few measurements prior to that time, they are compatible with the hypothesis that percentage fetal hemoglobin content is constant, at least past 182 days of gestation, and approximates 95 per cent of total hemoglobin. Though again the data are insufficient to demonstrate this without question, it is entirely possible that some decline begins prior to the two hundred fiftieth day of gestation. The apparent depression of the percentage content of fetal hemoglobin in the cord blood of babies involved in active erythroblastosis fetalis has been demonstrated many times since the report of Schulman and Smith. 51 Though only 3 cases of active erythroblastosis fetalis were studied in the present series, the percentage content of fetal hemoglobin from these bloods in no significant way deviates from the norm for the appropriate period of gestation. Though no significance can be attached to an observation based on 3 cases, it would suggest the need to repeat the fetal hemoglobin assay of cord blood in this disease by some method other than alkaline denaturation. The method at present is being reported in terms of preliminary findings. It might be considered to show promise in terms of the high order of reproducibility of repeat determinations and the low order of scatter as a function of the duration of gestation.
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