Hum. Genet. 53, 97-100 (1979) © by Springer-Verlag 1979

Improved Screening Test for Abnormal Hemoglobins from Dried Blood Samples K. Altland t* , M. Kaempfer 1, and H. Granda 2 Institut ftir Humangenetik, Universitfit Giessen, Schlangenzahl 14, D-6300 Giessen, Federal Republic of Germany 2Department of Medical Genetics, University of LaHabana, LaHabana, Cuba

Summary. A method is described wherin blood samples taken from adults or newborns and dried on filter paper can be used for hemoglobin analysis within 2 years after sampling. The samples are eluted in 8 M urea in the presence of 5% 2-mercaptoethanol and 2% of the neutral detergent Nonidet P-40. Then the individual a, fl, y, and d chains are separated by means of electrofocusing in 8 M urea-PAA gels. Up to 96 samples can be applied tO a gel using multiple syringes. Several hundred samples can be analyzed daily by one person. This method may be especially useful for preventive programs against sickle cell anemia as well as for h u m a n mutation monitoring systems.

Introduction In 1973, Heredero et al. (1974) described a high speed electrophoretic screening method for hemoglobin S, by which up to more than 1000 samples could be analyzed daily at a low cost. The aim of this work was to elaborate a test system to detect families that risk producing sicklemic offspring in populations with correspondingly elevated gene frequencies for H b S. This laboratory test, which worked well with fresh blood samples in a screening study of about 33,000 individuals (Granda et al., unpublished data) was completed by a modified solubility test which is of practical value to the practitioner (Heredero et al., 1976). The ready test solution supplied in ampules * To whom offprint requests should be sent used in this paper. PAA = polyacrylamide; PAG = polyacrylamide gel; TCA = trichloroacetic acid; PKU = phenylketonuria

Abbreviations

remained stable for up to 2 years, thus enabling almost untrained medical personnel to perform the test under almost any field conditions. Any diagnosis of heterozygosity for the sickle cell gene must be based on both a positive solubility test and an electrophoretic test. Thus, there remains the problem of how the practitioner in the countryside may complete his diagnostic procedure by electrophoretic examination of a blood specimen in a laboratory perhaps far away from his practice. In many countries, mailing samples in the usual way may result in delays of many days or weeks. In hot countries, blood samples may arrive in bad condition if they are not specially prepared. As demonstrated by Altland (1977), this problem can be solved by drying blood samples on filter paper and by using electrofocusing as an analytical procedure. The method is based on the separation of native hemoglobins. Tailing by partially denatured material was much less compared with the results of electrophoretic procedures. The identification of Hb A in the presence of H b F, however, was seen to be rather dependant on good charges of ampholytes, and the position of H b S was occasionally occupied by an undefined material of varying color intensity, thus increasing the risk of producing false positives. Using the method described in the present study, these drawbacks are reduced, since the analysis is performed on the level of the completely denatured globin chains.

Materials and Methods Capillary blood samples were collected from newborns in LaHabana by puncture of the heel, or from adults by puncture of the finger and then dried on filter paper, in a procedure similar to that used in mass screening systems for PKU. Samples

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K. Altland et al.: Improved Screening Test for Abnormal Hemoglobins

from some 20 individuals were sealed in a plastic bag together with silica gel and mailed to the laboratory in Giessen, where they arrived 4 weeks after blood collection. They were stored at +4°C for a period of 2 years or another 4 weeks until analysis without any special drying procedure. Fresh blood samples from the donors were used in the laboratory of LaHabana to determine the Hb phenotype by means of standard procedures such as the solubility test, PAG- and/or citrate-agar electrophoresis.

Preparation of Samples. Ninety-six samples of blood containing filter paper of 2.5 mm in diameter were placed in standard Ubottom Microtiter plates and eluted by adding 50 gl of 8 M urea with 5% 2-mercaptoethanol and 2% of the neutral detergant Nonidet P-40 (NP-40). After incubation for 1-3 h at 30°C, the samples were diluted by adding another 50-2001al of 5% 2mercaptoethanol in water. Then 10-20 gl of the diluted samples were applied to the electrofocusing gel.

Preparation of the Electrofocusing Gel. Two glass plates (265 x 200 × 1 ram) consecutively cleaned in deionized water, acetone, and etherwere separated by a U-frame of silicone rubber of 0.3 mm thickness, covered with 2 glass plates (265 x 200x4mm), and held together by steel clamps. Twenty milliliters of gel solution (T = 5; C = 3) were prepared from 9.6g of solid urea, 5.9ml of H20, 5 ml of a solution containing 19.4 g% (w/v) acrylamide, and 0.6 g% (w/v)bisacrylamide, 1 ml Servalytes 7-8 (Serva, Heidelberg) 20 gl of TEMED, and 200gl of 1.2g% (w/v) NH4-persulfate. The solution was deaerated in a vacuum, filled between the glass plates using a suitable plastic syringe, and covered with water. The polymerization time was adjusted to about 10 min at room temperature by appropriate variation of the amount of NH4-persulfate. After polymerization the glass cuvet with the gel was kept in a refrigerator for another 15 rain. The cuvet was opened with a knife and the gel with the adhering 1-mm glass plate was transferred to the cooling plate of the separation chamber (Desaphor or Mediphor, Desaga, Heidelberg) set at a temperature of 12-16°C.

Eleetrofocusing. An anodic wick (0.6 x 25.5 cm) of No. 17 Whatman paper was soaked in 0.5 M H3PO4 and put into the middle of the gel; then cathodic wicks soaked in 0.75 M NaOH were put on both long edges (25.5 cm) of the gel. Silicone rubber bands, as described by Altland (1977), with 24 slits (2 x 8.5 ram) or 48 holes of 4 mm in diameter were placed along a 1.5 cm distance from either side of the central anodic wick. The positive single electrode and a negative double electrode were put on the anodic and cathodic paper wicks and fixed with one or two glass plates (265 x 200 x 4 mm). A prerun was performed at 15 mA constant current, 850V limit, for 30rain. The samples were applied individually into the slits or by a 12-unit multiple syringe (Desaga, Heidelberg) into the holes of the silicone rubber bands and the run was continued for 3-5.5 h at 850 V constant voltage, 15 mA limit. Staining and Preservation. After separation the gels were fixed and stained by means of consecutive incubation for 15 min in the following solutions: (1) 20% w/v TCA; (2) tap water; (3) 9 vol. 0.3% w/v Coomassie R-250, filtered, 9 vol. ethanol, and 2 vol. acetic acid; (4) 3 vol. ethanol, 6 vol. water, and 1 vol. acetic acid; (5) 10% acetic acid, and 2% glycerol.

After photodocumentation the gel was dried overnight at room temperature between two sheets of cellophane fixed on a glass plate.

Adjustment of the pH Gradiant Produced by Servalytes 7-8. Commercially available Servalyte 7-8 contains considerable and varying amounts of ampholytes of p H < 7 and >8. To achieve the desired separation distances of globin chains without modifying electrode distance, unproper charges of Servalytes 7-8 were repurified in the following way: about 22 g of Sephadex G-25 (coarse) soaked in 100 ml of 10% (w/v) Servalyte 7-8 were applied to a cuvet (255 x 200 x l0 mm) with a bottom made of a 1-mm glass plate. Dry paper wicks (255 x 6mm) of No. 17 Whatman paper were placed on both long edges of the get, and the remaining supernatant solution was soaked by evenly strewing some dry Sephadex onto the surface of the gel. The electrodes were then put on the paper wicks and covered with a glass plate. Electrofocusing was performed in the Desaphor or Mediphor chamber (Desaga, Heidelberg) at 30mA constant current, 300V limit, overnight at 12-16°C. After termination of the run, the pH gradient was checked using a surface electrode (Ingold No. LOT 403-30-M8). The material within the range pH 6.6 to 8.5 was transferred to a column connected with a fraction collector and eluted with water. The fractions containing ampholytes were identified by means of conductivity measurement and pooled. A rough estimate of the concentration of purified ampholytes was made based on the volume of the pooled fractions, the fraction of the gel used for elution, and the total amount of ampholytes applied in the purification procedure. Since analytical electrofocusing proceeds at 2% (w/v) of the ampholytes, reconcentration after purification is not necessary. If wanted, for some reason, concentration may be performed by rotation evaporation at 40 ° C in the vacuum of a good water or membrane pump according to a suggestion of Radola (personal communication, 1979).

Separation of Alpha, Beta, and Delta Chains. For identification of the a, /?, and fi hemoglobin chains in the electrofocusing pattern, a and fl chains were separated by means of linear gradient column chromatography on CM-cellulose according to the procedure first described by Clegg et al. (1966). fl and chains were separated by means of preparative PAG electrophoresis of a normal hemolysate followed by electrophoretic elution of the A~ and A2 fraction using the PAG electrophoresis system Havanna (Desaga, Heidelberg) and the procedures described in the manual for that system.

Results T h e p r e p a r a t i v e s e p a r a t i o n o f a a n d fl g l o b i n c h a i n s b y m e a n s o f C M - c e l l u l o s e c h r o m a t o g r a p h y a n d the elect r o f o c u s i n g p a t t e r n s o f the f r a c t i o n a t e d e l u a t e are s h o w n in Fig. 1. It can b e seen t h a t the first p e a k obtained by chromatography is m i s s i n g m a t e r i a l p r e s e n t in t h e s e c o n d p e a k a n d t h a t the l a t t e r is m i s s i n g m a t e r i a l p r e s e n t in the t h i r d p e a k , w h i c h c o n t a i n s the fl g l o b i n chain. C o n v e r s e l y , the f r a c t i o n s o f t h e s e c o n d a n d the fl g l o b i n p e a k s h o w m a t e r i a l o f the p r e c e d i n g p e a k s in the e l e c t r o f o c u s i n g p a t t e r n . A similar o b s e r v a t i o n c a n be m a d e c o m p a r i n g t h e p r e -

K. Altland et al.: Improved Screening Test for Abnormal Hemoglobins

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Fig. l. Separation of a, fl, and g globin chains using column chromatography on CM cellulose (CM52, Whatman) via the method of Clegg et al. (1968) with minor modifications. The electrofocusing pattern obtained from each fraction is projected on the transmission curve of the eluate. The six dots on the abscissa label the fractions, whose patterns are repeated in Fig. 2. The third peak represents the fl chain and the last peak represents the a chain

Fig. 2. Electrofocusing patterns obtained from newborns of hemoglobin phenotype FA (no. 1, 4), FAS (no. 2), FAC (no.3), from adults of hemoglobin phenotype A (no. 5, 8, 9, 12, 19), AS (no. 6), AC (no. 7), from preparatively isolated Hb A1 (no. 10) and Hb A2 (no. 11), and from fractions obtained using column chromatography as demonstrated in Fig. 1 (nos. 13-18). The amount of samples corresponds to about 0.1 gl of full blood. Samples no. 1-8 were obtained from fnII blood dried on filter paper after mailing from LaHabana to Giessen at ambient temperature over a period of 4 weeks and storage for another 4 weeks at 4°C until elntion and analysis. The slight shift of the a and g chain zone toward the anode in sample no. 11 is due to a distortion of the pH gradient by high salt concentration in that sample. By comparison, it can be seen that the position of the beta s and betac zone is not occupied by other material a l p h a g l o b i n f r a c t i o n with the a g l o b i n f r a c t i o n . A z o n e which in the e l e c t r o f o c u s i n g p a t t e r n is i d e n t i c a l in p o s i t i o n with the c~ g l o b i n c h a i n is o b t a i n e d f r o m f r a c t i o n s o f the p o s t - b e t a g l o b i n region.

T h e p a t t e r n s o b t a i n e d f r o m dried b l o o d s p e c i m e n s 8 weeks after b l o o d c o l l e c t i o n are s h o w n in Fig. 2 t o g e t h e r with the p a t t e r n s o f f r a c t i o n s n u m b e r 1,4, 7, 13,21, a n d 24 f r o m c o l u m n c h r o m a t o g r a p h y (see

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K. Altland et al.: Improved Screening Test for Abnormal Hemoglobins

Fig. 1) and of the separated A1 and A2 hemoglobins obtained through PAG electrophoresis. The position of the alpha, beta A, beta s, beta c, gamma, and delta chains can be clearly identified by comparison of the patterns from the different sample sources indicated. All samples from adults, but also samples from many newborns show the delta zone, which can be seen in all samples from newborns when more material is applied. In several samples from adults (samples number 5, 8, 9, and 12, Fig. 2), a faint zone in the position of the g a m m a zone is visible which is absent in others (samples number 6 and 7, Fig. 2). The patterns obtained from blood specimens after 2 years of storage were identical in position with the major zones of the pattern obtained after 8 weeks of sample storage. They showed an increased background stain and different relative intensities of the zones. The reagents and plastic material consumed per 1000 samples, not including tile process of blood sampling and photodocumentation, have a value equivalent to about US $ 60.00. Two sets of 96 samples can be run simultaneously on the 40-cm cooling plate of the Desaphor electrophoresis chamber. With an appropriate amount of equipment, several hundered samples can be run daily in a small laboratory unit.

Discussion

The method described may be useful with regard to three aspects:

1. Mass Screening for Hemoglobin S in Preventive Programs Against Sickle Cell Anemia and Related Diseases. For collecting the capillary blood samples, donors will not have to go to a medical institution. Almost untrained medical workers can go to the donors at any place remote in distance and time from the laboratory facility and take the samples. In the case of screening for couples at risk, only about 2pq2 (for q = gene frequency of H b S and p = 1 - q ) of all couples will be composed of two heterozygotes for H b S and 2[2pq×(1-2pq)] of all couples will have one heterozygote. In both cases, the presence of Hb S must be confirmed or excluded by at least the solubility test. In the second case, the partner of the confirmed heterozygote for H b S must be checked for exclusion of a beta-thalassemia gene. Starting with a screening of samples collected on filter paper may present significant economic advantages. Furthermore, starting a screening with electrofocusing will be much more informative compared with a screening that starts with solubility testing,

So far, the method described here has not been tested with regard to the recognition of heterozygotes for beta-thalassemia, who may be recognized by their quantitatively elevated delta/beta-globin chain ratio.

2. Mass Screening in a Human Mutation Monitoring System. F r o m the patterns seen in Fig. 2, it follows that in newborns, charge variation of the a, fl, and ~ chain and perhaps also of the ~ chain (if more samples were applied) will be detectable using the method described. The fact that in m a n y countries blood specimens from all newborns are collected on filter paper within screening programs established for other reasons offers the possibility for determination of the actual mutation rate at the corresponding loci. Whether other nonhemoglobin gene loci can be checkedin the same way is subject to further investigation. 3. Prenatal Diagnosis. It is obvious that the separation obtained by electrofocusing of the individual globin chains and especially of the g a m m a and beta chain is much better than that obtained using column chromatography as described by Kan et al. (1972) for the prenatal diagnosis of sickle cell anemia and betathalassemia. Radioactive globin chains obtained by incubation of fetal red cells with amino acids of high specific radioactivity may be detected using autoradiography of the gels or may be quantitated by counting in a liquid scintillation counter after cutting out the stained individual zones and solubilizing the gel fractions. Much smaller amounts of sample would be necessary and many diagnoses could be performed simultaneously with a simpler and more economic method.

References

Altland, K.: Screening for abnormal hemoglobins in the newborn: A highly economic procedure using isoelectric focusing. In: Electrofocusing and isotachophoresis, pp. 295--301. B. J. Radola, D. Graesslin (eds.). Berlin-New York: Walter de Gruyter 1977 Clegg, B. J., Naughton, D. G,, Weatherall, D. J.: Abnormal human hemoglobins: Separation and characterization of the alpha- and beta-chains by chromatography and determination of two new variants, Hb Chesapeake and Hb J (Bangkok). Biochim. Biophys. Acta 19, 91--108 (1966) Heredero, L., Granda, H., Dortic6s, A.: A new form of the solubility test for hemoglobin S; results from a survey of 3000 cases. Clin. Chim. Acta 71, 515--519 (1976) Heredero, L., Granda, H., Suarez Aguilar, J. A., Altland, K.: An economic high speed electrophoretic screening system for hemoglobin S and other proteins. Hum. Genet. 21,167--177 (1974) Kan, Y. W., Dozy, A. M., Alter, B. P., Frigoletto, F. D., Nathan, D. G.: Detection of the sickle cell gene in human fetus. N. Engl. J. Med. 287, 1--5 (1972) Received July 18, 1979