Detection of z-Globin Chains in the Cord Blood byELISA (Enzyme-Linked Immunosorbent Assay): RapidScreening for a-Thalassemia 1 (Southeast Asian Type)

Ruchanee Ausavarungnirun, 1 Pranee Winichagoon, 2 Suthat Fucharoen, 2* Nava Epstein, 3 andRonald Simkins 4

1Department of Pathology, Faculty of Medicine, Srinakharinwirote University, Bangkok, Thailand2Thalassemia Research Center, Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital and Institute of

Science and Technology for Research and Development, Mahidol University, Bangkok, Thailand3Israel Institute for Biological Research, Ness-Ziona, Israel

4ISOLAB Inc., Akron, Ohio

Fetuses with homozygous a-thalassemia 1, in which the deletion of all four a-globingenes results in the absence of any a-globin chains, are severely anemic with clinicalfeatures of hydrops fetalis. Definitive diagnosis of a-thalassemia 1 carriers is difficultsince there are few red cell abnormalities. Recently Chui et al. found that minute amountsof embryonic z-globin chains are present in adult hemoglobin of the Southeast Asian typeof a-thalassemia 1 carriers.

In this study, we screened 521 cord bloods for a-thalassemia 1. Hemoglobin analysis,including quantitation of Hb Bart’s, was performed using the automated HPLC, a-thalassemia short program (VARIANT, Bio-Rad, Hercules, CA). Of these, 200 cord bloodsamples in which Hb Bart’s was demonstrated were tested for the presence of z-globinchains by ELISA using labeled anti- z monoclonal antibody. z-Globin ranged between 0.21and 0.83% in 19 specimens carrying a-thalassemia 1 gene. In the remaining 90 out of 109specimens in which Hb Bart’s was greater than 1.2%, z-globin was less than 0.17%. DNAanalysis revealed the presence of normal a-genotype and other types of a-thalassemiaincluding a-thalassemia 2 and Hb Constant Spring. One false positive was found in whichthe z-globin was 0.25% by ELISA but in which PCR indicated an a-thalassemia 2 hetero-zygote. Ninety-one samples with Hb Bart’s of less than 1.2% by HPLC are most likelynormal with a z-globin range between 0 and 0.14%. This study also showed that thefrequency of a-thalassemia 1 in Bangkok is 3.65%. Am. J. Hematol. 57:283–286, 1998.© 1998 Wiley-Liss, Inc.

Key words: zeta-globin antibody; ELISA; a-thalassemia 1

INTRODUCTION

During human embryonic and fetal development, anorderly change occurs in hemoglobin (Hb) production.The earliest embryonic Hbs are Hb Gower 1 (z2 e2), HbGower 2 (a2e2), and Hb Portland I (z2 g2). During thesecond and third trimesters of gestation, fetal Hb (a2 g2)becomes the dominant Hb until shortly after birth, whenit is gradually replaced by Hb A (a2b2) [1].

z-Globin chains, thea-globin-like chains found in theembryo and fetus, are generally believed to be presentonly during the first 2 months of gestation [2]. However,when all foura-globin genes are deleted, as in the HbBart’s hydrops fetalis syndrome,z-globin chains con-

tinue to be synthesized even in the third trimester ofgestation. Recently, minute amounts ofz-globin chainswere shown to be present in hemolysates from adult in-dividuals witha-thalassemia 1 due to the (−−SEA/) dele-tion [3]. A novel anti-humanz-globin chain monoclonal

Contract grant sponsor: Prajadhipok Rambhai Barni Foundation.

*Correspondence to: Suthat Fucharoen, MD, Thalassemia ResearchCenter, Institute of Science and Technology for Research and Devel-opment, Mahidol University, Salaya Campus, Nakorn-Phatom, Thai-land.

Received for publication 10 April 1997; Accepted 12 November 1997

American Journal of Hematology 57:283–286 (1998)

© 1998 Wiley-Liss, Inc.

antibody has been reported to detecta-thalassemia 1 in aslot blot immunobinding assay [4].

In general, thea-thalassemia trait is difficult to rec-ognize. Individuals who have decreased red cell osmoticfragility, low MCV, and slightly abnormal red cell mor-phology with normal HbA2 levels are suspected of beinga-thalassemia 1 carriers. Neonatal diagnosis ofa-thalassemia carriers may be possible by determination ofthe proportion of Hb Bart’s in cord blood. Definitivediagnosis ofa-thalassemia requires DNA analysis. Inthis study, a sensitive and specific enzyme-linked immu-nosorbent assay (ELISA) for human embryonicz-globinchains was used to detecta-thalassemia in newborn cordblood samples.

MATERIALS AND METHODS

Cord blood samples (521) were randomly collected bydirect aspiration from the umbilical vessels at the deliv-ery room of the Department of Obstetrics and Gynecol-ogy, Siriraj Hospital, Bangkok, Thailand. Hemolysateswere prepared from packed red cells for Hb analysis andELISA. Hemoglobin types and quantitation of Hb Bart’swere performed by the automated HPLC,a-thalassemiashort program (VARIANT, Bio-Rad, Hercules, CA).

Monoclonal Antibodies to z-Globin Chains

Monoclonal antibodies were generated in BalbC miceto purified z-globin chains. The antibodies did not reactwith hemoglobins A, S, F, C, and E. Antibodies werepurified from ascites on Protein G columns. The purifiedantibodies were conjugated to horseradish peroxidase us-ing a meta-periodate technique.

ELISA for z-Globin Chains

A standard curve consisting of 0, 0.05, 0.1, 0.2, 0.4,0.6, and 0.8%z-globin, was prepared from normal bloodhemolysate mixed with hemolysate from a Hb Bart’shydrops fetalis sample in which the amount ofz-globinwas determined by HPLC. A portion (10ml) of eachsample was diluted in 100ml of 50 mM Tris HCl, 0.15 MNaCl, pH 7.5, and incubated at 37°C for 30 min to allowthe z-globin in hemolysate to bind to the surface of theCostar (Cambridge, MA) microtiter plate. After rinsingwith TS, 100ml of a 1:4,000 dilution of 3E10-HRP con-jugate (horseradish peroxidase conjugated anti-z anti-body, ISOLAB Inc., Akron, Ohio) in 1% bovine serumalbumin in TST (Tris-Saline-Tween 20) was added andallowed to react at 37°C for 60 min. After rinsing, 100mlof the substrate TMB (3, 38, 5, 58,tetramethylbenzidine)was added, allowed to react for 5 min, and stopped by theaddition of 100ml of 0.2 M sulfuric acid. The absorbanceat 450 nm was measured with an ELISA plate reader.The z content of the samples was calculated from thestandard curve run with each batch of samples.

PCR techniques to determine thea-thalassemia 1 [5],a-thalassemia 2 (both −a3.7 and −a4.2 types) [6], and HbConstant Spring [7] were carried out with DNA extractedfrom buffy coat cells.

RESULTS

Hb Bart’s was detected in 200 out of 521 cord bloodsamples by the automated HPLC. Quantitation ofz-globin chains was carried out by ELISA in the samplespositive for Hb Bart’s. Figure 1 shows thez-globin con-tent and the level of Hb Bart’s in each individual. Ofthese, 109 had levels of Hb Bart’s greater than 1.2% asdetermined by HPLC. Table I shows the amounts ofz-globin chains as determined by ELISA. Elevatedamounts ofz-globin were detected in 19 out of 200 speci-mens. PCR technique confirmed the presence ofa-thalassemia 1 genotype in this group. There were two Hb

Fig. 1. Detection of a-thalassemia 1 by anti- z-globin anti-body in 200 cord blood. With combination of the measure-ment of Hb Bart’s by HPLC and z-globin by ELISA, the dataappear to fall into four groups. (1) Hb Bart’s <1.2% and z-globin <0.14%; normal ( aa/aa); (2) Hb Bart’s >1.2% and z-globin <0.17%; normal ( aa/aa) or a-thalassemia 2 heterozy-gote (− a/aa) or a-thalassemia 2 homozygote (− a/−a) or HbCS heterozygote ( aCSa/aa); (3) Hb Bart’s 6–11% and z-globin 0.21–0.83%; a-thalassemia 1 heterozygote (−−/ aa);(4) Hb Bart’s >15% and z-globin 0.3–0.6%; Hb H disease(−−/−a or −−/aCSa). j, Individuals carrying an a-thalas-semia-1 gene; h, no a-thalassemia-1 gene.

284 Ausavarungnirun et al.

H disease specimens (a-thalassemia 1/a-thalassemia 2)that hadz-globins 0.34 and 0.57% with Hb Bart’s of 24.2and 25.1%, respectively. There was one sample eachwith AE Bart’s disease (a-thalassemia 1/a-thalassemia2-EA) and EF Bart’s disease (a-thalassemia 1/a-thalassemia 2-EE) in whichz-globin was 0.42 and0.31%, respectively, with Hb Bart’s of 27.9 and 17.5%,respectively. The remaining 15 specimens werea-thalassemia 1 heterozygotes in which thez-globinsranged between 0.21 to 0.83% (mean ± SD4 0.57 ±0.21%) whereas the range of Hb Bart’s was 6 to 11%.This study also showed that the gene frequency ofa-thalassemia 1 in Bangkok is 3.65%.

Ninety out of the 109 specimens in which Hb Bart’swas greater than 1.2% had either normala-globin orother types ofa-thalassemia. All hadz-globins less than0.17% (mean ± SD4 0.04 ± 0.06%). Among these,three were double heterozygotes fora-thalassemia 2 andHb Constant Spring (Hb Bart’s was 9.2, 9.3, and 9.4%),one Hb Constant Spring homozygote (Hb Bart’s was15%), and 12 homozygotes fora-thalassemia 2 (HbBart’s 3.9–10%). The remaining were either heterozy-gotes fora-thalassemia 2 (Hb Bart’s was 1.8 ± 0.5%). HbConstant Spring (Hb Bart’s was 2.6 ± 0.47%), or normal(Hb Bart’s was 0.7 ± 0.37%) (Fig. 1). Those in which HbBart’s was less than 1.2% (91 cases) most likely had thenormal phenotype. In these casesz-globins ranged from0 to 0.14% (mean ± SD4 0.01 ± 0.03%). One false-positive sample with Hb Bart’s 1.9% and az-globin of0.25% by ELISA was found, which PCR indicated wasan a-thalassemia 2 heterozygote.

DISCUSSION

Infants with hemoglobin Bart’s hydrops fetalis syn-drome (homozygousa-thalassemia 1), with the deletionof all four a-globin genes, are stillborn or die shortlyafter birth. Pregnancies involving Hb Bart’s hydropsfetalis syndrome are associated with an increased riskof maternal complications, such as hydramnios, pre-eclampsia, antepartum or post-partum hemorrhage, anddifficult vaginal delivery [8,9]. There is also considerable

emotional strain for the mothers and their immediatefamily members. Prenatal diagnosis for Hb Bart’s hy-drops fetalis syndrome is possible by means of Southernblotting or polymerase chain reaction by using DNA ex-tracted from fetal cell samples, obtained by either chori-onic villi biopsy or amniocentesis [10–13]. However, theidentification of couples at risk of conceiving fetusesafflicted with Hb Bart’s hydrops fetalis syndrome isproblematic, and is presently dependent on the history ofbirth of a previous hydropic fetus.

Recently, Chui et al. and Luo et al. have found thatminute amounts of embryonicz-globin chains were pres-ent in adult carriers of the Southeast Asian type ofa-thalassemia 1 [3,4]. In the current study, we have dem-onstrated that a much simpler enzyme-linked immuno-sorbent assay (ELISA) using the horseradish peroxidaseconjugated anti-z antibody (3E10-HRP), can similarlydetectz-globin chains, and can be used as a diagnostictest to detect carriers ofa-thalassemia 1 due to the(−−SEA/) deletion. Previously we succeeded in using thisapproach to detectz-globin chains in specimens from 44individuals carrying thea-thalassemia 1 gene, whereasnone of the 80 normal individuals had detectablez-globin chains [14]. The data obtained in this study, witha combination of the measurement of Hb Bart’s byHPLC andz-globin by ELISA, appear to fall into fourgroups: (1) Hb Bart’s <1.2% andz-globin <0.14% (mean± SD 4 0.01 ± 0.03%); normal (aa/aa); (2) Hb Bart’s>1.2% andz-globin <0.17% (mean ± SD4 0.04 ±0.06%); normal (aa/aa) or a-thalassemia 2 heterozygote(−a/aa) or a-thalassemia 2 homozygote (−a/−a) or HbCS heterozygote (aCSa/aa); (3) Hb Bart’s 6–11% andz-globin 0.21–0.83% (mean ± SD4 0.57 ± 0.21%);a-thalassemia 1 heterozygote (−−/aa); (4) Hb Bart’s>15% andz-globin 0.3–0.6%; Hb H disease (−−/−a or−−/aCSa). The levels ofz-globin in each group of sub-jects at different gestational ages of pregnancy were alsoassessed but they did not seem to be related to prematu-rity (data not shown).

The gene frequency of the (−−SEA/) deletion in South-east Asia and Southern China is approximately 3%[8,15]. The current study has shown that the frequency ofa-thalassemia 1 in Bangkok is 3.65%, which is in agree-ment with that previously reported [15]. Homozygousa-thalassemia 1 containing the (−−SEA/) deletion is themajor cause of hydrops fetalis syndrome in this part ofthe world. The World Health Organization estimated in1983 that there were probably 20,000 infants born annu-ally afflicted with homozygousa-thalassemia 1 [16]. Thesimple ELISA described here is suitable for screeninglarge populations for carriers of the (−−SEA/) deletion.The sensitivity and specificity of this test was 100 and99.5%, respectively (Table II). The general applicationof this simple screening test for carriers ofa-thalasse-mia 1 with the (−−SEA/) deletion in Southeast Asia and

TABLE I. Amounts of z-Globin Chains Detected by ELISATechnique in 200 Samples With Positive Hb Bart’s by theAutomating HPLC

No.

z-Globin chains (%)

Range Mean ± SD

Normal 91 0–0.14 0.01 ± 0.03a-Thalassemia 1 15 0.21–0.83 0.57 ± 0.21Hb H Disease 2 0.34, 0.57 —EA Bart’s 1 0.42 —EF Bart’s 1 0.31 —Othera-thalassemia 90 <0.17 0.04 ± 0.06

Detection of a-Thalassemia 1 by ELISA 285

Southern China may help to improve the genetic coun-seling and the quality of obstetrical care provided tothose women at risk of conceiving infants with Hb Bart’shydrops fetalis syndrome.

ACKNOWLEDGMENTS

This work was supported by the Prajadhipok RambhaiBarni Foundation, Bangkok, Thailand. We thank Dr.Louise D. Wadsworth (Department of Pathology, B.C.Childrens Hospital and University of British Columbia,Vancouver, Canada) for his comments on the manu-script.

REFERENCES

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2. Peschle C, Mavilio F, Cae A, Migliaccio G, Migliaccio AR, Salvo G,Samoggia P, Petti S, Guerriero R, Marinucci M, Lazzaro D, Russo G,Mastroberardino G: Haemoglobin switching in human embryos: Asyn-chrony of z->a and e->g globin switches in primitive and definitiveerythropoietic lineage. Nature 313:235, 1985.

3. Chui DHK, Wong SC, Chung S-W, Patterson M, Bhargava S, PoonM-C: Embryonicz-globin chains in adults: A marker fora-thalassemia1 haplotype due toa > 17.5 kb deletion. N Engl J Med 314:76, 1986.

4. Luo HY, Clarke BJ, Gauldie J, Patterson M, Liao S, Chui DHK: Anovel monoclonal antibody based on diagnostic test fora-thalassemia1 carrier due to the (−−SEA/) deletion. Blood 72:1589, 1988.

5. Winichagoon P, Fucharoen S, Kanokpongsakdi S, Fukumaki Y: De-tection ofa-thalassemia 1 (Southeast Asian type) and its applicationfor prenatal diagnosis. Clin Genet 47:318, 1995.

6. Baysal E, Huisman THJ: Detection of common deletionala-thalassemia-2 determinants by PCR. Am J Hematol 46:208, 1994.

7. Fucharoen Sp, Fucharoen G, Fukumaki Y: Simple non-radioactivemethod for detecting haemoglobin Constant Spring gene. Lancet 335:1527, 1990.

8. Liang, ST, Wong VCW, Zo WWK, Ma HK, Chan V, Todd D: Ho-mozygousa-thalassemia: Clinical presentation, diagnosis and man-agement. A review of 46 cases. Br J Obstet Gynecol 92:680, 1985.

9. Bowman E, Watts J, Burrows R, Chui DHK: Haemoglobin Bart’shydrops fetalis syndrome. Haematologia 20:125, 1987.

10. Chan V, Ghosh A, Chan TK, Wong V, Todd D: Prenatal diagnosis ofhomozygousa-thalassemia by direct DNA analysis of uncultured am-niotic fluid cells. Br Med J 288:1327, 1984.

11. Zeng Y-T, Huang G-Z:a-Globin gene organization and prenatal di-agnosis ofa-thalassemia in Chinese. Lancet i:304, 1985.

12. Rubin EM, Kan YW: A simple sensitive prenatal test for hydropsfetalis caused bya-thalassemia. Lancet i:75, 1985.

13. Chehab FF, Doherty M, Cai S, Kan YW, Cooper S, Rubin EM: De-tection of sickle cell anaemias and thalassaemias. Nature 329:293,1987.

14. Ausavarungnirun R, Winichagoon P, Epstein N, Simkins R, FucharoenS: Detection ofz-globin chains, a marker for Southeast Asian type ofa-thalassemia 1 by enzyme-linked immunosorbent assay (ELISA).Thai J Hematol Transfusion Med 6:185, 1996.

15. Fucharoen S, Winichagoon P: Hemoglobinopathies is Southeast Asia.Hemoglobin 11:65, 1987.

16. WHO Working Group: Community control of hereditary anaemias:Memorandum from a WHO meeting. Bull WHO 61:63, 1983.

TABLE II. Sensitivity and Specificity of ELISA Technique inthe Detection of z-Globin Chains in Individual Cord BloodContaining a-Thalassemia 1*

Samples witha-thal 1 (−−SEA)

Samples withouta-thal 1 (−−SEA) Total

Negative 0 180 180Positive 19 1 20Total 19 181 200

*Sensitivity 419

19× 100 4 100%; Specificity4

180

181× 100 4 99.4%.

286 Ausavarungnirun et al.