Comp. Biochem. Physiol., 1977, Vol. 58 B, pp. 103 to 107. Pergamon ,ares& Priated tn Great Britain

UTILIZATION OF A DIFFERENTIAL HEMOGLOBIN ELUTION PROCEDURE AS A RAPID ASSAY FOR

INDENTIFICATION OF EMBRYONIC AND ADULT CHICKEN RED BLOOD CELLS

KIMBERLY KLINE AND BOB G. SANDERS The University of Texas, Zoology Department, Austin, TX 78712, U.S.A.

(Received 5 January 1977)

Al~tract--l. A hemoglobin elution-staining procedure has been developed for distinguishing embryonic chick red blood cells from adult chicken red blood cells.

2. Adult hemoglobin is eluted from red blood cells with 1.9 M potassium phosphate buffer, pH 7.2; whereas, embryonic hemoglobin is retained within the cells and gives positive staining with erythro- sin B.

3. The hemoglobin elution-staining pattern during development can be correlated with two embryonic hemoglobins as detected by polyacrylamide gel electrophoresis.

4. The series of red blood cells staining with erythrosin B correspond to the primary erythrocyte series suggesting that hemoglobin expression during development is correlated with different cell popula- tions.

INTRODUCTION

The expression of gene products specific for various stages of embryonic development is well documented for several proteins, with the hemoglobins being prominent. In addition to their importance for studies of regulation during development, embryonic gene products take on an added significance with the observations of Gold & Freedman (1965) and Sanders & Kline (1975) in that embryonic markers which are lost with development reappear in cancerous adult animals.

Our laboratory is in the process of identifying oneo-developmental markers which disappear with development and reappear in the cancerous chicken. Certain red blood cell membrane antigens behave as onto-developmental markers (Sanders & Kline, 1975). Therefore, it was of interest to examine other red blood cell developmental markers, namely hemo- globins. The hemoglobins of chickens have been stud- led by electrophoretic and immunological techniques as to the types of hemoglobins and to their time of appearance and disappearance during development in the laboratories of Fraser et al. (1972), Schalekamp et al. (1972) and Brown & Ingram (1974). There appears to be at least six embryonic hemoglobins and two adult hemoglobins. Structural analyses and iden- tification of the polypeptide chains of the multiple hemoglobins of chickens remain to be done before one can conclusively determine the number of genes involved. However, based on the immunological and electrophoretic studies of Schalekamp et al. (1972), seven genes coding for hemoglobin synthesis are pos- tulated to be expressed at some time during the life- time of chickens.

It has been of interest in our laboratory to develop

techniques for investigating red blood cell markers at an individual cell level. Thus, the technique of dif- ferential elution of hemoglobins from embryonic and adult human red blood cells described by Kleihaner & Betke (1958, 1963) and more recently by Kabat (1974) were investigated as a means of developing a rapid method for screening chicken blood smears for cells containing embryonic hemoglobins.

In this paper we report on an elution-staining tech- nique which permits one to distinguish red blood cells obtained from adult and certain aged embryonic chickens. The elution-staining technique was used to follow the hemoglobin profile during embryonic de- velopment and to correlate the elution-staining profile to embryonic hemoglobins and to the primary eryth- rocyte series.

MATERIALS AND METHODS

Elution-stainino procedure

Blood was collected in heparin (Sodium salt of heparin obtained from Organon, Inc., 1000 USP units/ml were used. Diluent = 0.75% saline) from outbred white leghorn chicken embryos (obtained locally) by dissecting surround- ing tissues from a vein and collecting the blood with a disposable pasteur pipette. Blood from adult outbred leg- horn chickens (obtained locally) was obtained by wing venepuncture. Blood cells collected in Alsever's solution or Na2EDTA were unsatisfactory for the elution-staining technique.

These blood smears were prepared on microscope slides and permitted to dry at room temperature. After drying, the sfides were stored for ca. 24 hr prior to elution and staining procedures. Slides (aged 24 hr) were immersed for 3--5 rain in 1.9 M potassium phosphate buffer, pH 7.2 (51.7gin KH2PO, (anhydrous)/200ml distilled water; 165.5 g K=HPO, (anhydrous)/500 ml distilled water. Com- bining 168 ml 1.9 M KH2PO4 plus 432 ml 1.9 M K2HPO 4 should give a pH of 7.2. We found it necessary to adjust the pH down with ca. 17ml of 1.gM KH2PO4) at

This work was supported by DHEW, PHS, National Cancer Institute Grant Number CA12851 (B.G.S.)

103

1 0 4 K I M B E R L Y K L I N E A ~ D B O B G . S A N D E R S

24-25':C. The slides were agitated gently during extraction. Following extraction, the slides were transferred immedi- ately to 95% ethanol (completely denatured Mallinckrodt Chemical Works) for 10 rain at 24-25°C, rinsed with dis- tilled water for 5 sec and then immersed for 5 rain in meth- anol (Baker analyzed reagent--J. T. Baker Chemical Co.) at 24-25°C. The slides were then rinsed with distilled water and stained for 4min with 0.1~o aqueous Erythrosin B (Fisher Scientific Co.) at 25°C. After staining, the smears were thoroughly rinsed with distilled water and allowed to air dry then examined microscopically at 450 × magnifi- cation.

Identification of red blood cell series

Blood smears from different developmental stages of embyronic development were stained with standard Jen- ner May-Grunwald-Giemsa staining procedures cited by Ewald (1976). The stained smears were examined at 1000X magnification and erythrocyte series identification was based on descriptions by Lucas & Jamroz (1961).

tlemoylobin preparation and analyses

Red blood cells were washed three times in 0.75,°.,0 NaCI. An equal volume of distilled water was added to the packed red blood cells and the tube containing the red blood cell-water mixture was dipped in a dry ice-acetone mixture for rapid freezing. Upon thawing, the contents

/ .

. , . .

~.2 h.

Fig. 1. Dried smears of chncken red blood cells [aged 24 hrb were treated with 1.9 M potassium phosphate, pH 7.2 buffer at 25~C for hemoblobin elution. The smears wcrc fixed in 95°0 ethanol for 10min and absolute methanol for 5 min. The eluted, fixed smears were stained for the presence of hemoglobin with I"o Erythrosin B. Red blood cells from a 5 day chick embryo are depicted in Fig. la Red blood cells from a 6 day chick embryo are depicted in Fig. lb. Red blood cells from an adult chicken arc depicted in Fig. lc. Magnification for photographs is 450 x.

were centrifuged and the hemoglobin was decanted. Cyan- methemoglobin preparations and concentrations wcrc made and determined by the standard procedures cited by Crosby et al. (1954).

Equal concentrations of cyanmethemoglobtn prep- arations made from red blood cells of differing embryonic stages were separated with a BioRad Model 220 vertical polyacrylamide slab gel electrophoresis apparatus using the anionic buffering system of Davis (1964). Gels were stained with 0.2°,0 3,3-dimethoxybenzidine/50,, glacial acetic acid aqueous solution for 30min and destained with 1 I. methanohl 1. H20:200ml acetic acid destain solution for comparative purposes.

R E S U L T S

Differential elution-staining results arc depicted in Fig. la, b, c. Fig. la shows the staining pat tern obtained with 5 day embryonic red blood cells. All the red blood cells stained pink. Fig. lb shows the staining pat tern ~btained with 6 day chick embryo red blood cells. The staining was heterogeneous in that cells stained from pink to dark red. A low per- centage of non-stained cells are seen. Fig. l c shows the typical "ghosts" or non-staining pat tern obtained with red blood cells obta ined from adult chickens.

In the course of this study three variables in- fluenced the hemoglobin elution-staining results dra- matically and had to be controlled before obta ining repeatable data. The variables were aging of the red blood cell smears prior to elution-staining, molarity of elution buffer, and the temperature at which the elution-staining procedure was carried out. The effects of these three variables on embryonic and adult red blood cells are depicted in Table 1. Permit t ing blood

Utilization of a differential hemoglobin elution procedure

Table 1. Effects of molarity, temperature, and aging of blood smears on elution-staining procedures

105

Potassium phosphate molarity Temperature* Aging of blood Red blood cell pH 7.2 (°C) smears* (hr)

smears 1.6 1.7 1.8 1.9 2.0 2.1 2.2 20 25 30 2 24 over 48

Embryonic (2-10 days) - + + + + + + + + + - + +

Adult . . . . + + + - - ( + / - ) - - +

* Temperature and aging effects were assayed at 1.9 M potassium phosphate, pH 7.2 buffer only. Positive staining is represented by + and non-staining is represented by - .

smears to age for 24 hr, utilizing 1.9 M potassium phosphate buffer at pH 7.2 for hemoglobin elution, and maintaining a constant 24-25°C temperature throughout the assay proved to be the best conditions for obtaining hemoglobin differences in individual red blood cells obtained from embryonic and adult chickens.

Table 2 depicts red blood cell changes associated with various ages of embryonic development, namely percent primary erythrocytes and percent cells stain- ing with Erythrosin B after elution. The elution-stain- ing profile was as follows. Uniform pink stained cells were found on the earliest day tested, day 2, and stained cells were found through day 9 of embryonic development. After the 9th day of embryonic develop- ment the red blood cells behave as adult red blood cells, namely all cells were non-staining ghosts. Cells stain a uniform pink color on days 2 through day 5. Day 6 shows 80~o of the cells staining, 45~ of which are pink and 35~ dark red. Of the staining cells on days 7, 8, and 9 only the dark red stained cell type was encountered.

The elution-staining patterns obtained in these studies were compared with hemoglobin (Fig. 2) and erythrocyte changes (Table 2 and Fig. 2) occurring with development. The hemoglobin elution-staining pattern closely parallels the presence and subsequent absence of embryonic hemoglobins E3 and E4 (Fig. 2 and Fig. 3).

Furthermore, the phasing of the hemoglobins E 3 and E4 as well as the elution-staining pattern corre- sponded to primary erythrocyte series which de- creases with development (Table 2).

DISCUSSION

Genetically coded fetal-adult differences such as the hemoglobins serve as markers for studies of gene regulation and are useful as markers in determining whether embryonic gene products are expressed in the diseased state. Hemoglobins obtained from hemo- lysates of embryonic and adult red blood cells can be identified using the various chemical and immuno- logical methods described by Fraser et al. (1972), Schalekamp et al. (1972) and Brown & Ingrain (1974). However, such assays do not permit the distinction between individual red blood cells containing embryonic and adult hemoglobins.

Elution of hemoglobins from red blood cells is based primarily on the solubility properties of the hemoglobins at a particular salt concentration and pH. In our studies chicken embryonic hemoglobins were found to be less soluble than chicken adult hemoglobins at 1.9 M potassium phosphate, pH 7.2; since the embryonic chick cells retained their hemo- globins during 1.9 M potassium phosphate, pH 7.2 elution and could subsequently be stained with a stain specific for hemoglobin, namely Erythrosin B. Hemo- globin is eluted from adult chicken red blood cells and the cells appear as ghosts upon staining.

The elution-staining data described in this paper is repeatable only when temperature, molarity, and red blood cell aging is controlled. The hemoglobin changes occurring during the aging of red blood smears are unclear. Perhaps oxidation of the proteins continue several hours after the cell smears have dried.

Table 2. Red blood cell changes associated with embryonic chicken development

Age of embryonic Percent Total percentage Percent

chicken primary of red blood non-stained (days) erythrocytes cells staining (Ghosts)

2 100 100 0 3 --* 100 0 4 100 100 0 5 93 96 4 6 86 80 20 7 46 33 67 8 15 20 80 9 8 10 90

10 2 0 100

* Not tested.

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DAYS EMBRYONIC CHICK DEVELOPMENT

Fig. 2. The elution-staining pattern obtained with red blood cells from developing chick embryos is compared with the cell type and hemoglobin profile of similar aged chick embryos. The elution-staining data is recorded as the % cells staining and is depicted as . . . . . The % primary erythrocytes are depicted as . . . . . . The % of E3 + E4 hemoglobin types were taken from Schalekamp et al.

(1972) and Fig. 3 and are depicted by - - -

The question as to which hemoglobin(s) is staining in the embryonic cells is important in view of the observations by Fraser et al. (1972), Schalekamp (1972), and Brown & lngram (1974) that chickens have multiple hemoglobins. The elution-staining curve parallels the presence and subsequent loss of the E 3 and E4 hemoglobins suggesting that the stain- ing of embryonic cells is correlated with one, the other, or perhaps both embryonic hemoglobins. E 3 and E4 hemoglobins are the major hemoglobins present in red blood cells of embryos 2-5 days of age (Figs 2 and 3). Adult hemoglobins do not appear until day 6 or 7 (Fig. 3). The changes in erythrocyte

series with development (Table 2 and Fig. 2) appear to have a direct correlation with the elution-staining pattern. The elution-staining procedure appears to correlate with the hemoglobin types produced in the primary erythrocyte series and not for the hemo- globin types produced in the secondary erythrocyte series.

The specificity of this elution-staining pattern for embryonic hemoglobins of the primary erythrocyte series may prove useful in examining normal erythro- poiesis versus erythropoiesis during different disease states.

SUMMARY

A hemoglobin elution procedure is described for differentiating primary and secondary red blood cell series in chicken blood smears. Potassium phosphate buffer (1.9 M, pH 7.2) treatment of blood smears results in the elution of hemoglobin from red blood cells of adult chickens and the retention of hemo- globin in the red blood cells obtained from early stages of embryonic development. Staining of the treated blood smears with Erythrosin B reveals pink to red embryonic cells whereas non-stained red blood cells (ghosts) are obtained from chickens greater than 10 days embryonic development. The hemoglobin elu- tion-staining pattern parallels the presence and sub- sequent absence of two embryonic hemoglobins as well as the loss of the primary erythrocyte series, sug- gesting that hemoglobin expression during develop- ment is correlated with different cell populations.

The elution-staining procedure provides a rapid sensitive screening method for distinguishing primary erythrocytes from adult erythrocytes which can in conjunction with other embryonic markers be used in studies on gene regulation in embryonic and dis- eased states.

Acknowledgements--The authors wish to acknowledge the excellent technical assistance of Cynthia J. Morton.

+

A 4 6 8 I0 A Fig. 3. Electrophoretic comparison of hemoglobins obtained from different stages of chick embryonic develop- ment and adult chickens. Hemoglobin was obtained from packed, red blood cells of adult (A) and days 4, 6, 8, and I0 of embryonic chick development. The electrophoretic procedures are given in the paper. Hemoglobin concen- trations as determined by optical density were adjusted so that each well received identical concentrations. The adult hemoglobin band closest to the cathodal region is A 2 and the hemoglobin band migrating to the anodal pole is At. The embryonic hemoglobins can be distinguished best on days 4 and 6. E4 is closest to the cathodal pole.

The most intense band represents hemoglobin E3.

REFERENCES

BROWN J. L. & INGRAM V. M. (1974) Structural studies on chick embryonic hemoglobins. J. biol. Chem. 249, 3960-3972.

CROSBY W. H., MUNN J. I. 8,L Fua'rH F. W. (1954) U.S. Armed Forces Med. J. 5, 693-698.

DAVIS B. J. (1964) Disc electrophoresis--ll. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121, 404-427.

EWALD S. J. (1976) Studies of the Fc receptor on the sur- face of chicken lymphoid cells. Ph.D. Dissertation. The University of Texas at Austin, 124-126.

FRASER R., HORTOrq B., DuPOURQUE D. 8/. CHERNOFF A. (1972) The multiple hemoglobins of the chick embryo. Cell. Physiol. 80, 79-87.

GOLD P. & FREEDMAN S. O. (1965) Specific carcinoem- bryonic antigens and the human digestive system. J. exp. Meal 122, 467-481.

KABAT D. (1974) An elution procedure for visualization of adult hemoglobins in human blood smears. Blood 43, 239-242.

KLEIHAUER E 8,~ BETKE K. (1958) Felaler und bleibender blutfarbstoff in erythrocyten und erythroblasten yon menschlichen feten und neugeborench, Blur 4, 241-249.

Utilization of a differential hemoglobin elution procedure 107

KLEIHAUER E. & BETrd~ D. (1963) Elution procedure for demonstration of methaemoglobin in red blood cells of human blood smears. Nature, Lond. 199, 1196-1197.

Lucks A. M. & J~oa~oz C. (1961) Atlas of avian Hemat- ology. Washington, USDA, pp. 104-140.

SANDERS B. G. & KLINE K. (1975) Fetal-tumor membrane

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Scnla,E~o4P M., SC'nAL~KXUP M., VAN GOOR D. & SLINGERLAND R. (1972) Re-evaluation of the presence of multiple hemoglobins during the ontogenesis of the chicken. J. Embryol. exp. Morph. 28, 681-731.