Objectives: This study aimed to evaluate the UBI MAGIWELTM ζ-GLOBIN ELISA Kit for the presumptive diagnosis of αo-thalassaemia. The ELISA results obtained were confirmed by molecular characterisation of αo-thalassaemia using a Duplex-PCR. Methods: Routine peripheral blood counts and red cell indices were determined in 94 blood samples sent for Hb analysis. Hb subtypes were quantified by high performance liquid chromatography (HPLC) and Hb electrophoresis conducted on agarose gel at pH 8.5. Zeta-globin chain levels were determined using the UBI MAGIWELTM ζ-GLOBIN ELISA Kit. Molecular analysis was performed using a duplex-PCR which simultaneously amplifies a normal 136 bp sequence between the ψα−α2-globin genes and a 730 bp Southeast Asian deletion-specific sequence (–SEA) between the ψα2−θ1-globin genes. Results: Using the ELISA assay kit, 20 blood samples were presumptively identified as α-thalassaemia carriers from elevated ζ-globin chains (OD>0.3) while the remaining 74 blood samples showed OD<0.3. Molecular characterisation confirmed amplification of the 136 bp normal fragment in all the blood samples. Seventeen of the 20 DNA samples from the α-thalassaemia carriers amplified the SEA-deletion specific fragment. The remaining three DNA samples did not amplify the SEA-deletion specific fragment but amplified the normal α-globin gene sequence, indicating the presence of amplifiable DNA in these samples. Further molecular characterisation of the α-3.7 and -α4.2 deletions and Hb Constant Spring (CS) mutation showed the absence of these defects in these 3 DNA samples. Conclusion: This preliminary investigation showed the sensitivity and specificity of the UBI MAGIWEL ζ-globin ELISA kit as 100 % and 96.1 % respectively in the detection of α-thalassaemia-1 carriers (–SEA)

George, Elizabeth

Hassan, R.

Lim, E. J.

Tan, K. L.

Tan, Mary Anne Jin Ai

Wee, Y. C.

Zakaria, Z.

Universiti Putra Malaysia Institutional Repository

Identification of   αo-Thalassemia (--SEA ) Using  ELISA for Embroyonic Zeta-globin Chain Detection  81Identification of αo-thalassaemia (–SEA) Using an Enzyme-Linked Immunosorbent Assay (ELISA) for EmbryonicZeta-globin Chain Detection – A Preliminary Study1JAMA Tan, 2E George, 3EJ Lim, 4Z Zakaria, 5R Hassan, 1YC Wee &1KL Tan1Department of Molecular Medicine, Faculty of Medicine, University of Malaya,50603 Kuala Lumpur2Department of Clinical Laboratory Sciences, Faculty of Medicine and HealthSciences, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia3BP Laboratories, Kuala Lumpur, Malaysia4Department of Medicine, Institute of Medical Research, Kuala Lumpur5Department of Pathology, Hospital Kuala Lumpur, MalaysiaABSTRACTObjectives: This study aimed to evaluate the UBI MAGIWELTM ζ-GLOBIN ELISA Kitfor the presumptive diagnosis of αo-thalassaemia.  The ELISA results obtained wereconfirmed by molecular characterisation of αo-thalassaemia using a Duplex-PCR.  Methods:Routine peripheral blood counts and red cell indices were determined in 94 blood samplessent for Hb analysis.  Hb subtypes were quantified by high performance liquidchromatography (HPLC) and Hb electrophoresis conducted on agarose gel at pH 8.5.Zeta-globin chain levels were determined using the UBI MAGIWELTM ζ-GLOBIN ELISAKit.  Molecular analysis was performed using a duplex-PCR which simultaneously amplifiesa normal 136 bp sequence between the ψα−α2-globin genes and a 730 bp Southeast Asiandeletion-specific sequence (–SEA) between the ψα2−θ1-globin genes.  Results: Using theELISA assay kit, 20 blood samples were presumptively identified as α-thalassaemia carriersfrom elevated ζ-globin chains (OD>0.3) while the remaining 74 blood samples showedOD<0.3.  Molecular characterisation confirmed amplification of the 136 bp normal fragmentin all the blood samples. Seventeen of the 20 DNA samples from the α-thalassaemiacarriers amplified the SEA-deletion specific fragment.  The remaining three DNA samplesdid not amplify the SEA-deletion specific fragment but amplified the normal α-globin genesequence, indicating the presence of amplifiable DNA in these samples.  Further molecularcharacterisation of the α-3.7 and -α4.2 deletions and Hb Constant Spring (CS) mutationshowed the absence of these defects in these 3 DNA samples. Conclusion:  This preliminaryinvestigation showed the sensitivity and specificity of the UBI MAGIWEL ζ-globinELISA kit as 100 % and 96.1 % respectively in the detection of α-thalassaemia-1 carriers(–SEA).Keywords: ELISA, ζ-globin chains, α-thalassaemia-1, Duplex-PCR, SoutheastAsian deletionCorresponding Author: JAMA TanE-mail: jamatan@hotmail.comMalaysi n Journal of Medicine nd Health Sciences Vol. 2(2) June 2006 : 81-87Malaysian Journal of Medicine and Health Sciences Vol. 2 (2) June  2006 82   JAMA Tan, E George, EJ Lim,  Z Zakaria, R Hassan, YC Wee & KL TanINTRODUCTIONThalassaemia is a genetic blood disorder of haemoglobin synthesis and is the most commoninherited haematological disorder in Malaysia.  Thalassaemia is a public health problem inMalaysia as the incidence of α-thalassaemia carriers among the Malays and Chinese isapproximately 3-4 %.[1]  However, the majority of these α-thalassaemia carriers are unawareof  their  thalassaemia  status  as α-thalassaemia-1 or  α0 (–/aa) and  α-thalassaemia-2 or α+(-α/αα) are asymptomatic and difficult to detect haematologically.Alpha-thalassaemia is caused by deletions or mutations within the α-globin genecomplex and this leads to a decrease or an absence of α-globin chain production. Alpha-thalassaemia can result from a deletion of either one (α+-thalassaemia) or both (α0-thalassaemia) α-globin genes.[2]  The most severe form of α-thalassaemia is Hb Bart’shydrops foetalis, a condition that is not compatible with life.  Hb Bart’s hydrops foetalis iscommon in Southeast Asia [3] and is responsible for 60-90 % of Bart’s hydrops foetalis casesin this region.[4]  The estimated frequency of α-thalassaemia carriers in Southeast Asia isapproximately 3-5 % [5] and the Southeast Asian deletion (–SEA) is the most common type ofdeletion involved.[6]  The –SEA deletion of 17.5 kb along the α-globin gene involves thedeletion of the ψα2-, ψα1-, α2-, α1-and θ1-genes leaving only the ζ-genes intact (Fig.1).[7, 8]Figure 1.  Southeast Asian deletion in the α-globin gene clusterNote: Genes are represented as solid boxes, pseudogenes as dotted boxes and hypervariable regionsas zigzag lines. The α-globin gene cluster on chromosome 16 is arranged in order telomere - ζ2,ψζ1, ψα2, ψα1, α2, α1, and θ1 – centromere. The Southeast Asian defect (–SEA) of 17.5 kbdeletes the ψα2, ψα1, α2, α1, and θ1 – globin genes and spares the ζ2, ψζ1 – globin genes.There is an urgent need to identify carriers of αo-thalassaemia in couples with the SEA-deletion as they are at risk of producing a gestation with Hb Bart’s hydrops foetalis.Molecular characterisation and prenatal diagnosis for α-thalassaemia in Malaysia arecurrently carried out using a Duplex-PCR.[9]  The duplex-PCR simultaneously amplifies boththe normal sequence between the ψα−α2-globin genes and the SEA-specific deletion in asingle PCR reaction, and thus offers a rapid and specific prenatal diagnosis method for α-thalassaemia in the Malaysian population.Adult carriers of αo-thalassaemia with the Southeast Asian deletion (–SEA) have beenreported to possess increased levels (0.01-1%) of embryonic ζ-globin chains in theirerythrocytes.[10,11] The elevated embryonic ζ-globin chains can be detected using   Malaysian Journal of Medicine and Health Sciences Vol. 2 (2) June 2006Identification of αo-Thalassemia (--SEA ) Using  ELISA for Embroyonic Zeta-globin Chain Detection  83fluorescence microscopy after immunocytological staining of erythrocytes with anti-humanζ-globin chain antibodies.[12, 13]  A simpler ζ-globin chain immunosorbent assay (ELISA)was subsequently developed to detect αo-thalassaemia carriers with the –SEA deletion [14, 15]and sensitivity of the ζ-globin ELISA assay was reported at 89.3% and specificity at 100%in a study carried out using peripheral blood.[16]  The ζ-globin ELISA assay offers a simpleand cost-effective diagnostic method for the identification of carriers of (–SEA) deletional αo-thalassaemia.  In addition, this method can be used in diagnostic laboratories whereequipment and trained staff for molecular diagnosis are not available.  The UBI MAGIWELTM Zeta Globin (ZAM) Qualitative Test kit (United Biotech, Mountain View, California, USA)detects αo-thalassaemia carriers with the (–SEA) deletion and α-thalassaemia-1 carriers fromother α-thalassaemia mutations that spare the embryonic ζ-globin genes and causes tracesof ζ-peptides to persist throughout life.This project is a preliminary study to evaluate the UBI MAGIWEL TM ζ-globin ELISA kitfor the presumptive diagnosis of  αo-thalassaemia carriers with the (–SEA) deletion.  Theresults obtained from the ELISA assay were compared with molecular studies using theDuplex-PCR to confirm the SEA deletion in αo-thalassaemia carriers.METHODSPatient SamplesThree laboratories in the Klang Valley entered 94 (30, 30, 34 per site respectively) bloodsamples. Entrance criteria were Hb analysis testing ordered.  The EDTA anticoagulatedwhole blood samples were used for automated blood counts, to quantify Hb subtypes byhigh performance liquid chromatography (HPLC)  for UBI MAGIWEL TM ζ-globin ELISA kitanalysis and DNA studies.  Routine peripheral blood counts and red cell indices weredetermined according to standard laboratory procedures.  Hb analysis was performed on allblood samples with a MCV <80 fl and MCH <27 pg.  Hb subtypes were quantified by HPLCusing the Variant Hb Analyser (Bio-Rad Laboratories, Hercules, California, USA).  AutomatedHb electrophoresis was conducted on agarose gel at pH 8.5 (Sebia Hydrasys).UBI MAGIWEL TM ζ-globin ELISA KitThe UBI MAGIWELTM Zeta Globullin (ZAM) Qualitative Test (United Biotech, MountainView, California, USA) is a solid phase enzyme-linked immunosorbent assay (ELISA) thatprovides screening for elevated ζ-globin levels in whole blood.  The ELISA was performedfollowing  manufacturer’s instructions.  Patient’s blood samples (100 µ l) were mixed with200 sample diluent (200 µ l). 100 µ l of the treated blood sample were dispensed into microplatewells coated with immobilised antibodies. After incubation at 37oC for 60 minutes, the wellswere washed five times with washing buffer.  Enzyme conjugate was then added and thewells incubated for another 30 minutes at 37oC before another 5 washes with washingbuffer.  Two solutions (A and B) were mixed and then added to the wells for 15 minutes atroom temperature.  The reaction was stopped with the addition of 50 ml of 1N H2SO4.  Opticaldensity was read at Absorbance 450 using a microplate reader. A positive colour (yellow)reaction (OD>0.3) indicates the presence of ζ-globin chain peptides in the blood sample. AMalaysian Journal of Medicine and Health Sciences Vol. 2 (2) June  2006 84   JAMA Tan, E George, EJ Lim,  Z Zakaria, R Hassan, YC Wee & KL Tansample is negative when the OD reading is less than 0.2. Positive (blood from α-thalassaemia-1 patients with the (–SEA) deletion) and negative (blood from normal individuals) controlblood and blank wells were included in every ELISA assay.DNA ExtractionDNA was extracted using proteinase K and sodium dodecyl sulphate.  Extracted DNA waspurified by phenol-chloroform and precipitated with ethanol.  Precipitated DNA was washedin alcohol, air-dried and then solubilised in double distilled water.  DNA concentration andpurity were measured spectrophotometrically.Duplex-PCRThe duplex-PCR was performed using primers SEA1: 5’-CTCTGTGTTCTCAGTATTGGAGG-3’and SEA2: 5’-ATATATGGGTCTGGAAGTGTATC-3’ to amplify the 730 bp SEA deletion-specific sequence.  Primers α1: 5’-TACTGTAGATACCCGTGTACAA-3’ and α2: 5’-ATCATGATGGAAACATAGTAAT-3’[17]  were used to amplify the 136 bp normal sequencebetween the ψα−α2-globin genes.  The PCR cycling conditions were initial denaturation at95°C for 5 minutes followed by 30 cycles of denaturation at 93°C for 1 minute, annealing at50°C for 1 minute, synthesis at 72°C for 3 minutes and a final extension at 72°C for 6 minutes.DNA from a known heterozygote for α-thalassaemia (–SEA/αα), a homozygous αo-thalassaemia (–SEA/–SEA) and a normal individual (αα/αα) were amplified concurrently inevery PCR to act as positive and negative controls.Gel Electrophoresis of PCR ProductsAmplified PCR products were resolved by electrophoresis on 1.5% agarose gels.Electrophoresis was carried out at 90 V for 45 minutes.  DNA bands were observed usingultraviolet light irradiation after ethidium bromide staining and were recorded using a geldocumentation system.RESULTSOut of the 94 blood samples sent for Hb analysis, 20 were presumptively identified as αo-thalassaemia carriers by peripheral blood counts, red cell indices, HPLC analysis and Hbelectrophoresis.   The 20 samples consisted of 14 Chinese and 6 Malay patients.  Using theUBI MAGIWEL TM ζ-globin ELISA kit, all 20 samples showed elevated ζ-globin chains(OD>0.3).  The OD reading of the remaining blood samples were <0.3.Using the duplex PCR, the 136 bp normal ψα−α2-globin gene sequence was confirmedin all blood samples.  The SEA-deletion specific sequence was not amplified from DNA inthose with an OD reading <0.3.  Seventeen out of the 20 DNA samples from αo-thalassaemiacarriers with elevated ζ-globin chains amplified the SEA-deletion specific sequence.  ThreeDNA samples did not amplify the SEA-deletion but amplified the normal 136 bp ψα−α2-globin gene sequence, indicating the presence of amplifiable DNA in these samples .   Malaysian Journal of Medicine and Health Sciences Vol. 2 (2) June 2006Identification of αo-Thalassemia (--SEA ) Using  ELISA for Embroyonic Zeta-globin Chain Detection  85Table 1.   Molecular characterisation of DNA samplesSample Ethnic A450 136 bp ψα−α2- —SEA α3.7 -α4.2 HbCSnumber group globin genesequence1219 Chinese 1.648 Positive Negative Negative Negative Negative1208 Malay 0.850 Positive Negative Negative Negative Negative1209 Malay 0.987 Positive Negative Negative Negative NegativeFurther molecular characterisation of the α-globin gene showed the absence of the -α3.7 and -α4.2 deletions in these three samples (Table 1).  In addition, Hb Constant Spring dueto a T-C bp substitution in the termination codon of the α-2 globin gene was also notpresent in the three DNA samples.On comparing the ELISA method with molecular characterisation using Duplex-PCR,the ELISA method was found to have a sensitivity of 100 % and a specificity of 96.1 %.DISCUSSIONMolecular characterisation using the duplex-PCR was used to evaluate the ELISAimmunological technique for the detection of αo-thalassaemia carriers with the SoutheastAsian deletion.  The ELISA kit used was the UBI MAGIWEL TM ζ-globin ELISA kit thatdetects αo-thalassaemia carriers resulting from the SEA deletion and α-thalassaemia-1 carriersfrom other α-thalassaemia mutations that spare the embryonic ζ-globin genes and causestraces of ζ-peptides to persist throughout life.  Results from this study showed the sensitivityand specificity of the ELISA kit as 100% and 96.1 % respectively, when using an OD cut-offvalue of 0.3 as determined by the manufacturers.  In a study of an ELISA method for ζ-globinchain detection for αo-thalassaemia carriers with the SEA deletion where a cut-off value of0.2 was used, the investigators reported the sensitivity and specificity of the ELISA to be96.4 % and 98.2 % respectively.16Three of the blood samples with elevated ζ-globin chains detected in this study did notamplify the SEA deletion-specific sequence (Table 1). In a study of 200 cord bloods for α-thalassaemia-1 using both ELISA and PCR techniques, one false positive was reportedwhere the ζ-chains were elevated but PCR confirmed the patient as an α-thalassaemia-2heterozygote.18 The ELISA method was also reported to be unable to detect the SEA deletionin some cases of Hb H disease blood samples. 16  The investigators reported that incompletelysis of hypochromic microcytic cells together with the low erythrocyte count in Hb Hpatients may have produced a smaller quantity of ζ-globin chains  in  the  blood  lysate  andthus,  produced  the  false-negative  result.Alpha-thalassaemia with two α-globin gene deletions (–MED) with elevated ζ-globinchains and positive ELISA results have been reported.11,16 However, molecularcharacterisation of  the Mediterranean gene deletion was not carried out in the three DNAsamples that were negative for the SEA deletion in this study. Molecular characterisation ofthe single α-gene deletions (-α3.7 and -α4.2) and the non-deletional α-thalassaemia (HbConstant Spring) was carried out in the three  DNA  samples.  The  samples were found tobe  negative for these three gene defects. 86   Jama Tan, E George, EJ Lim,  Z Zakaria, R Hassan, YC Wee & KL TanHb Bart’s hydrops foetalis observed in the majority of hydropic pregnancies in theMalaysian Chinese results from the inheritance of the Southeast Asian deletion from bothparents.  Thus, a simple test for the confirmation of the double α-globin gene deletion inboth chromosomes is sufficient for the antenatal diagnosis of Hb Bart’s hydrops foetalis inMalaysia.  The duplex-PCR allows the detection of the Southeast Asian deletion and thenormal ψα-α2-globin gene sequence in a single PCR reaction.  It is a rapid, sensitive andspecific method. However, equipment for molecular analysis and trained staff is required.The UBI MAGIWEL TM ζ-globin ELISA kit is a rapid assay and does not require sophisticatedequipment or highly trained staff.  This preliminary investigation showed sensitivity andspecificity of the UBI MAGIWEL TM ζ-globin ELISA kit as 100 % and 96.1 % respectively.The sample numbers involved in this study are small and a larger study is needed todocument the specificity of the ELISA method in the detection of the SEA deletion in αo-thalassemia carriers.ACKNOWLEDGEMENTWe thank Amrina, Fadzlina, Mohd Sufi, Rossalyna and Safarina for skilled laboratoryassistance with the ELISA  tests to identify ζ-globin chains.REFERENCES[1] George E.  Beta-thalasaemia major in Malaysia; an on-going public health problem.  Med JMalaysia 2001; 56, 4: 397-400.[2] Thalassaemia International Federation. Prevention of thalassaemias and other Haemoglobindisorders. Vol  2: Laboratory Methods. Nicosia, Cyprus, 2005.[3] Wong HB.  Prevention of thalassaemias in South-East Asia. Ann Acad Med 1985; 14(4): 654-665.[4] Chui DHK, Waye JS.  Hydrops fetalis caused by α-thalassaemia: an emerging health careproblem. Blood 1998; 91: 2213-2222.[5] Todd D, Lai MCS, Braga CA, Soo HN.  Alpha-thalassaemia in Chinese: cord blood studies.  BritJ Haematol 1969; 16: 551-556.[6] Ko TM  Hsieh FJ  Hsu PM  Lee TY.  Molecular characterisation of severe α-thalassaemiascausing hydrops foetalis in Taiwan.  Am J Med Genet 1991; 39: 317-20.[7] Bowden DK, Vickers MA, Higgs DR.  A PCR-based strategy to detect the common severedeterminants of α-thalassaemia.  Brit J Haematol 1992; 82: 104-108[8] Kattamis AC, Camaschella C, Sivera P. Surrey S, Fortina P.  Human α-thalassaemia syndromes:detection of molecular defects.  Am J Haematology 1996 ; 53: 81-91[9] YC Wee, KL Tan, PC Tan, SF Yap, JAMA Tan.  Rapid and cost effective prenatal diagnosis ofhaemoglobin Bart’s Hydrops Foetalis Syndrome using a Duplex-Polymerase Chain Reaction.Malaysia Journal of Medicine 2005 ; 60: 447-453.Identification of   αo-Thalassemia (--SEA ) Using  ELISA for Embroyonic Zeta-globin Chain Detection  87[10] Chui DHK, Wong SC, Chung SW, Patterson M, Bhargava S, Poon MC.  Embryonic ζ-globinchains in adults: a marker for α-thalassaemia-1haplotype due to a >17.5kb deletion.  N Engl JMedicine 1986;314: 76-79.[11] Tang W, Luo HY, Albitar M, Patterson M, Eng B, Wayne JS, Liebhaber SA, Higgs Dr, ChuiDHK.   Human  embryonic  ζ-globin  chain  expression in deletional α-thalassaemias. Blood1992; 80: 517-522[12] Tang W, Luo HY, Eng B, Wayne JS, Chui DHK.  Immunological test to detect carriers of (–SEA)deletional α-thalassaemia.  Lancet 1993; 342: 1145-1147.[13] Chan LC, So JCC, Chui DHK. Comparison of haemoglobin H inclusion bodies with  ζ-globinin screening for α-thalassaemia.  J Clin Path 1995;48: 861-864.[14] Simkins RA, Than KA, Schapiro B, Chooi ES, Daoust PR.  Correlation of ζ-globin ELISAwith PCR for (–SEA) deletion and clinical diagnosis for -thal-1 trait.  Ann NY Acad Sci 1998;850: 429-431.[15} Lafferty J, Crowther M, Wayne JS, Chui DHK.  A reliable screening test to identify adultcarriers of the (–SEA) alphao –thalassaemia deletion.  Detection of embryonic zeta-globinchains by enzyme-linked immunosorbent assay.  Am J Clin Path 2000; 114: 927-931.[16] Ma SK, Ma V, Chan AYY, Chan LC, Chui DHK.  Routine screening of (–SEA) α-thalassaemiadeletion by an enzyme-linked immunosorbent assay for embryonic ζ-globin chains.  ActaHaematol 2002; 108: 8-12.[17] Chehab  FF, Doherty M, Cai  SP,  Kan YW, Cooper  S,  Rubin EM.  Detection of sickle cellanaemia and thalassaemias. Nature 1987;329: 293-294.[18] Ausavarungnirum R, Winichagoon P, Fucharoen S, Epstein N, Simkins R.  Detection of zeta-globin chains in the cord blood by ELISA (enzyme-linked immunosorbent assay): rapidscreening for alpha-thalassaemia 1 (Southeast Asian type).  Am J Hematol 1998 ; 57(4): 283-286

Identification of α°-thalassaemia (–SEA) using an enzyme-linked immunosorbent assay (ELISA) for embryonic zeta-globin chain detection – a preliminary study

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Identification of α°-thalassaemia (–SEA) using an enzyme-linked immunosorbent assay (ELISA) for embryonic zeta-globin chain detection – a preliminary study

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