Hb I-High Wycombe [β59(E3)Lys→Glu]: The First Instance in Japan

15,661 cord bloods from Jamaican infants were examined for abnormal hemoglobins wine alkaline cellulose acetate electro-phoresis for the initial screening, supplemented by acid agar gel electrophoresis for samples exhibiting abnormal hemoglobin bands. Of the 16 electrophoretic variants which were detected, six were fully characterized and found to be: four Hb F Port Royal (α₂^Gγ₂125 Glu→Ala) and two Hb F Victoria Jubilee (α₂^Aγ₂80 Asp→Tyr). The Hb F Port Royal samples each constituted about one eighth of the total Hb F as did seven additional samples presumed to be Hb F Port Royal. The infants with this variant exhibited no special hematological characteristics or other consistent associations. Both Hb F Victoria Jubilee samples occurred in somewhat lower proportions of the total Hb F compared with Hb F Port Royal and exhibited an apparent increase of free alpha chains in the whole hemolysate. The data available on detectable gamma chain variants suggest that a specific point mutation may occur in either a Hb_Gγ or a Hb_Aγ locus.     

The synthesis of the Gγ and Aγ chains of human fetal hemoglobin in erythroid colonies cultured from peripheral blood BFUe's of normal adults and newborn and of subjects with an Aγ or a Gγ chain abnormal fetal hemoglobin

Peripheral blood mononuclear cells from normal adults, normal newborn infants, and from newborn and adult subjects with one of three γ chain variants (^GγF-Malta I, ^GγF-Port Royal, ^AγF-Hull) were cultured in vitro with erythropoietin. The ³⁵S-methionine-labelled hemoglobin from 13- to 15-day-old BFUe-derived colonies was studied by chromatography on columns of DEAE-cellulose and the quantities of Hbs A₂, F_x, and F₀ determined. The percentages of ^Gγ and ^Aγ chains in isolated Hb F_x and Hb F₀ were determined using high-performance liquid chromatography (HPLC) of the tryptic peptides of these proteins. Calculation of these percentages was based on the total activities of the ^GγT-15 and ^AγT-15 peptides which contain one (³⁵S-labelled) methionyl residue each and can be separated by the HPLC procedure. The data show an increased synthesis of Hb F in the ?adult”? colonies and a decreased synthesis in the ?newborn”? colonies. The ^Gγ to ^Aγ ratio of the Hb F from adult colonies varied greatly. The percentages of ^Gγ and ^Aγ chains in the Hb F from adult colonies correlated with the percentages in the Hb F isolated from the Hb F of circulating red blood cells. The ^Gγ to ^Aγ ratio in the Hb F from newborn colonies was high as in the Hb F from cord blood samples. ^Gγ and ^Aγ chain abnormal Hb F variants were readily detectable in colonies from both adults and newborn. The ^Gγ to ^Aγ ratio in the Hb F of colonies of adult Hb F-Malta I and Hb F-Hull heterozygotes approached 1, but that of adult Hb F-Port Royal heterozygotes remained about as high as in colonies from newborn heterozygotes. The percent Hb F-Port Royal in the Hb F of adult colonies was twice that in the Hb F of newborn colonies. These results are discussed in the light of information from recent detailed studies of genomic DNA assuming controlling functions for the segments of DNA that are interspersed between the various structural genes.     

Presumptive diagnosis of common haemoglobinopathies in Southeast Asia using a capillary electrophoresis system

Introduction: This study was conducted to examine ability of the Capillarys 2 haemoglobin (Hb) testing system to assist in presumptive diagnosis of common Hb variants found in Southeast Asia including five α‐chain and nine β‐chain variants. Methods: Blood samples with unknown Hb variants sent from other hospitals to our centre for identification were re‐analysed using the Capillarys 2 Hb analyser (SEBIA). DNA analyses by allele specific PCR assays were used to establish the final diagnoses. Results: Five α‐globin chain variants including Hbs Q‐Thailand, Queens, Siam, Constant Spring and Paksé were detected. In heterozygous form, the machine demonstrated clearly two abnormal derivatives of Hbs A and A₂ for the former three variants. Small peaks of Hb Constant Spring and Hb Paksé were observed but could not be differentiated. In contrast, only one abnormal peak of Hb A was observed for β‐globin chain variants including those with more negative charge (Hb J‐Bangkok, Hb Hope and Hb Pyrgos) and less negative charge (Hb D‐Punjab, Hb S, Hb Korle‐Bu and Hb C). Hb Tak, an elongated β‐chain variant was co‐separated with Hb F whereas the Hb Malay co‐migrated with Hb A in a subject with high Hb A₂β‐ thalassaemia trait. Conclusion: The capillary electrophoresis system could clearly demonstrate the presence of abnormal Hbs and provide useful information for presumptive diagnoses in most cases. The Hb analysis results could help in selection of appropriate DNA testing for final diagnoses of these variants.     

alpha Chain and gamma chain abnormal hemoglobins in newborn babies: structural and genetic aspects

Structural studies and quantitative analyses were conducted on the hemoglobin of 55 newborn babies. Seven alpha chain variants (G-Philadelphia, Montgomery, Inkster, I-Philadelphia, Matsue-Oki, Winnipeg, and O-Indonesia) were present in 26 heterozygous newborns (17 black, eight Caucasian, and one Indonesian). The relative amount of the alpha X containing abnormal Hb F of the Hb G-Philadelphia and Hb Winnipeg babies was less than observed in heterozygous adults, which may indicate a decreased rate of assembly of the alpha X-gamma dimer over that of the alpha X-beta dimer. Of the 29 newborns with gamma chain variants, 16 were Caucasian babies; of these 15 had a Hb A gamma F-Hull heterozygosity and one a Hb G gamma F-Marietta heterozygosity. Six black babies were heterozygous for Hb A gamma F-Texas-I and six for Hb G gamma F-Port Royal. One Japanese baby had a heterozygosity for A gamma F-Iwata and a second was heterozygous for A gamma TF-Yamaguchi. Quantitative analyses of the isolated normal Hb Fo as well as an evaluation of the relative amounts of the Hb Fx in the red cell lysates gave data useful for a speculation of the genetic condition in each of these babies. It was concluded that the babies with the Hbs F-Texas-I, F-Iwata, F-Hull, and F-Marietta were simple heterozygotes with either the G gamma x A gamma/G gamma x A gamma X or the G gamma x A gamma/G gamma X x A gamma genic arrangement. The babies with Hb F-Port Royal had a G gamma x G gamma X/G gamma x A gamma arrangement, which may result from a (to be determined) gene conversion. The newborn baby with Hb F-Yamaguchi has the G gamma x A gamma x/A gamma T-X.-. genic arrangement, suggesting the presence of three distinctly different gamma chain genes of which one, the A gamma T-X gene, produces an A gamma chain (with threonyl at position gamma 75 and an Asn at position gamma 80) at a level usually seen for G gamma rather than A gamma chains. These studies were greatly facilitated by the use of high pressure liquid chromatographic methods.     

Hemoglobin British Columbia (alpha2beta2 101(G3)Glu replaced by Lys). A new variant with high oxygen affinity.

Hemoglobin British Columbia was found in an East Indian living in Vancouver, British Columbia, Canada. Its structure was demonstrated to be alpha2beta2 101(G3)Glu replaced by Lys. It has a significant increase in oxygen affinity, decrease in heme-heme interaction, but normal Bohr effect. Unlike Hb Rush (beta101 Glu replaced by Glu), it is as stable as Hb A to heat and alcohol denaturation. By both cellulose acetate electrophoresis and chromatography the undissociated Hb British Columbia moves between Hb S and Hb A rather than behaving like Hb C. However, the dissociated abnormal beta chain behaves like beta C. The substitution is at the alpha2beta2 contact region. Except for a mild erythrocytosis, the propositus has normal hematological findings.     

Molecular basis and hematological features of hemoglobin variants in Southern Thailand

Hemoglobinopathy (abnormal hemoglobin or hemoglobin variant) is an inherited disorder that results in the abnormal structure of globin chains of the hemoglobin (Hb) molecule. Many abnormal Hbs have been characterized worldwide, including more than 20 variants in Thailand. The Bio-Rad Variant II HPLC system is used for investigating hemoglobin variants at Songklanagarind Hospital. This system has been shown to be a sensitive, specific, and reproducible method, but some hemoglobin variants such as Hb Tak and Hb D-Punjab cannot, as yet, be clearly separated by this method. The aim of this study was to investigate the prevalence of hemoglobinopathy in southern Thailand using DNA sequencing and study the severity of each hemoglobin variant. A total of 58 hemoglobin variant samples were obtained from blood samples undergoing routine hemoglobin typing at Songklanagarind Hospital. Genomic DNAs were extracted from the samples, and the globin genes were analyzed by using PCR-direct sequencing. The molecular analysis revealed eight hemoglobin variants: 28 Hb C, 12 Hb D-Punjab, 7 Hb Tak, 4 Hb G-Makassar, 2 Hb Lepore-Hollandia, 2 Hb Q-Thailand, 2 Hb O-Indonesia, and 1 Hb Hope. The distribution of hemoglobin variants in southern Thailand is associated with geographic and/or ethnic backgrounds. This study may help hematologists understand better the prevalence of hemoglobin variants and their hematological features in this region.     

Haemoglobin F Port Royal (α2Gγ2, 125Glu → Ala)

S ummary . A ^Gγ-chain variant, Hb F Port Royal, with an electrophoretic mobility intermediate between Hb S and Hb C, was found in a Jamaican-Negro infant, and made up 14–15% of the total Hb F. A glycinamidation procedure was employed to aid in determining the amino-acid residue substitution of γ^{125Glu → Ala}, and the presence of glycinc in position 136.     

Two new alpha chain variants: Hb Boghé [alpha58(E7)His-->Gln, alpha2], a variant on the distal histidine, and Hb CHarolles [alpha103(G10)His-Tyr, alpha1

The present paper reports two new alpha-globin chain variants: Hb Boghé [alpha58(E7)His-->Gln, alpha2] and Hb Charolles [alpha103(G10)His-->Tyr, alpha1]. Hb Boghé was found in a 12-month-old girl who was treated for malignant histiocytosis at 9 months of age and received a bone marrow transplant from her sister. Hb Boghé was undetectable by isoelectrofocusing and high performance liquid chromatography of hemoglobins. It was only revealed by polyacrylamide gel electrophoresis of globin chains in the presence of urea-Triton X-100 and accounted for 10% of the total hemoglobin. Hb Charolles was detected in a 46-year-old patient who presented with microcytosis and hypochromia. It was easily detected by isoelectrofocusing and high performance liquid chromatography. Hb Charolles accounted for 11% of the total hemoglobin. Characterization of the two hemoglobin variants was achieved by DNA and restriction enzyme analyses. Oxygen equilibrium curves measured on whole blood with Hb Boghé were normal. DNA sequencing revealed the association of Hb Charolles with a common mutation of the alpha2 polyadenylation site: AATAAA-->AATAAG.     

The different types of alpha-thalassemia: practical and genetic aspects

From May 1985 to October 1987, 1,564 Southeast Asians living in Hawaii were screened for hereditary anemias. Microcytosis was determined by electronic red cell indices and morphology; iron deficiency was ruled out by normal red cell distribution width and normal protoporphyrin levels; Hb E was determined by electrophoresis; beta-thalassemia (thal) heterozygotes were identified by raised Hb A2 on column chromatography. alpha-Thalassemia heterozygotes were diagnosed by exclusion. Family studies helped identify or confirm diagnoses, especially for the alpha-thal-2 heterozygotes (-alpha/alpha alpha) and homozygotes (-alpha/-alpha). Provisional diagnoses are being checked by DNA analyses. Iron deficiency prevented detection of possibly coexisting alpha-thalassemias in 97 individuals. Technical problems included the obscuring of standard criteria for recognizing the alpha-thal variants by the presence of Hb E or beta-thal. In such cases, alpha-thal could only be detected by family studies or DNA analyses. Problems with hemoglobin (Hb) electrophoresis included Hb H migrating beyond the edge of the strip if incubation was not closely monitored, and difficulty in detecting the small amounts of unstable Hb Constant Spring. DNA analyses also had limitations, since the nondeletion alpha-thalassemias would not be detected by routine Southern blotting. DNA analyses suggested that about 50% of presumed alpha-thalassemias were alpha-thal-2 (-alpha/alpha alpha) variants, and a corresponding number of alpha-thal-2 variants were among the apparent normals. Gene frequencies in the unselected Lao subjects were approximately 0.2 for Hb E, at least 0.1 for (-alpha), usually a rightward (alpha -3.7) type, 0.04 for (-), and 0.01 for a beta-thal. Multistep screening for the alpha- and beta-thalassemias was an effective and efficient strategy.     

Hb Isehara (or Hb Redondo) [beta 92 (F8) His----Asn]: an unstable variant with a proximal histidine substitution at the heme contact.

A 50-year-old Japanese female patient was found to have hemolytic anemia. Isoelectrofocusing of her hemolysate revealed two abnormal hemoglobin bands, one of which was very close to the Hb A2 band, and the other between the Hb A2 and Hb F bands. CM-cellulose column chromatography of the globin prepared from the abnormal hemoglobin showed that the abnormal chain eluted faster than the normal beta and delta chains; the beta X chain, however, did not separate from the normal beta chain in urea cellulose acetate electrophoresis. An instability test of the patient's hemolysate revealed the presence of an unstable component. Structural analysis of the abnormal beta chain indicated that the histidine residue at beta 92(F8) was replaced by an asparagine or aspartic acid residue. DNA amplified by polymerase chain reaction was sequenced by the dideoxy method. The nucleotide sequence of the beta 92 codon was AAC instead of CAC, suggesting that the amino acid substitution corresponded to His----Asn, which is the same as is found in Hb Redondo or beta 92(F8)His----Asn----Asp.     

Two new hemoglobin variants: Hb Brem-sur-Mer [beta9(A6)Ser-->Tyr] and Hb Passy [alpha81(F2)Ser-->Pro (alpha2)

Two new hemoglobin (Hb) variants: Hb Brem-sur-Mer [codon 9 (TCT-->TAT); beta9(A6)Ser-->Tyr] on the first exon of the beta-globin gene and Hb Passy [codon 81 (TCC-->CCC); alpha81(F2)Ser-->Pro (alpha2)] on the second exon of the alpha2-globin gene, are described. The two variants were characterized by DNA sequencing and mass spectrometry (MS). Hematological abnormalities: microcytosis and hypochromia were found only in the carrier of Hb Passy. In the absence of an association with an alpha-thalassemic deletion or mutation, the mutation 81(F2)Pro could induce a possible alpha-thalassemia (thal).     

Neonatal screening and mass-spectrometric analysis of hemoglobin variants in Japan

Using dried blood on filter paper, neonatal screening and mass-spectrometric analysis of hemoglobin variants were carried out in Osaka Prefecture, Japan. The following results emerged. (1) Of 140,597 newborns screened, 28 alpha (1/5,000) and 91 gamma (1/1,500) chain variants were detected. (2) Of 65 gamma chain variants examined, more than half (in 40 samples) were Hb F Yamaguchi (A gamma T 80 Asp----Asn). This fact explains the high incidence of gamma chain variants in the present study. (3) Two new gamma chain variants, Hb F Fuchu (G gamma 21 Glu----Gln) and Hb F Minoo (G gamma 72 Gly----Arg), were detected.     

Structural and functional studies of hemoglobin Moabit (alpha 86(F7) LeuArg

An abnormal hemoglobin fraction was detected on high performance liquid chromatography profile performed for the measurement of glycated hemoglobin in a 55-year-old caucasian patient. The structural and functional studies were performed by standard techniques. Separation of hemoglobins by alkaline electrophoresis and by IEF revealed a slightly more rapid fraction than does Hb S. By acid electrophoresis, no abnormal Hb fraction could be observed. Separation of globin chains by electrophoresis demonstrated an alpha-chain variant and by chromatography, a fraction which eluted between ß and gamma globin chains. Tryptic digests and amino acid analysis have demonstrated a previously described substitution of LeuArg alpha 86(F7).     

Hemoglobin Bougardirey-Mali beta 119 (GH2) Gly replaced by Val. An electrophoretically silent variant migrating in isoelectrofocusing as Hb F.

Hemoglobin Bougardirey-Mali was detected by isoelectrofocusing during a screening in a 32 years old African, a native of Mali. This abnormal Hb, representing 35% of the total, exhibited the same pI as that of Hb F. In contrast, it was indistinguishable from Hb A in all the electrophoretic systems tested, and equally by its resistance to alkaline denaturation. Structural studies have shown that the abnormality was localized on the beta chain. A fingerprint of the tryptic digest of the aminoethylated beta chain indicated the absence of the beta T12 b. The presence of an abnormal beta T12 b was suspected in the T14-15 spot, as indicated by the intensity of staining and its amino acid composition. beta T12 b was isolated by chromatography on PA 35. Its sequential analysis by manual Edman-dansyl degradation showed that glycine 119 was replaced by a valine residue. This mutation is localized in a alpha 1 beta 1 contact, which makes the molecules slightly unstable. The clinical consequences of this mutation seem to be minor; similar observations have been reported for the other Hb mutated at the same locus, i.e. Hb Fannin-Lubbock beta 119 Gly leads to Asp.     

Hb Bart's level in cord blood and deletions of alpha-globin genes

The white blood cell DNA of 36 cord blood samples with Hb Bart's in the red blood cells was studied for alpha-globin gene deletions by hybridization of DNA fragments digested by the restriction endonucleases Eco RI, Hpa I, Bam HI, and Bgl II. All 16 DNA samples from cord blood with Hb Bart's below 3% and no other abnormal hemoglobin had one alpha-globin gene deletion (alpha thal2), except one which had two alpha-globin gene deletions (alpha thal1). Most of the alpha thal2 were of the rightward deletion alpha thal2 genotype. Two new types of alpha thal2 variation was found, probably due to a polymorphism somewhere in an area outside the alpha-globin gene. All 14 cases with Hb Bart's between 3.5% and 8.5% and no other abnormal hemoglobin had two alpha-globin gene deletions (alpha thal1), except one that did not have any alpha-globin gene deletion and one that had one alpha-globin gene deletion. Three DNA samples of cord blood with Hb Bart's accompanied by Hb CoSp did not have any alpha-globin gene deletion. Sixty-five DNA samples from cord blood without Hb Bart's or other abnormal hemoglobin had no alpha-globin gene deletions, except one that had one alpha-globin gene deletion (alpha thal2). Two of the 65 DNA samples were found to have triplicated alpha-globin gene loci.     

Isoelectric focusing of human hemoglobin: its application to screening, to the characterization of 70 variants, and to the study of modified fractions of normal hemoglobins

Isoelectric focusing on slabs of acrylamide gel was adapted for the screening of abnormal hemoglobins, the characterization of 70 human variants, and the study of minor fractions of normal hemoglobin. The screening method was as fast and inexpensive as conventional techniques, allowed the simultaneous analysis of some 50 samples of whole blood, and yielded resolution superior to that obtained by other methods with hemolysates. Among the 70 variants, 31 mutants could not be separated from HbS by cellulose acetate electrophoresis. The characterization technique of electrofocusing allowed us to distinguish between most variants. Only one mutant, Hb Galveston, could be confused with HbS. Hb Koln, the most frequent unstable mutant, exhibited a special pattern. HbA1C was separated from HbA. Preliminary results indicate that quantitation of HbA1C by gel scanning is feasible.     

Distinct phenotypic expression associated with a new hyperunstable alpha globin variant (Hb heraklion, alpha1cd37(C2)Pro>0): comparison to other alpha-thalassemic hemoglobinopathies

Clinical phenotypes associated with abnormal globin chain biosynthesis may result in thalassemia (deficient quantity) or hemolytic anemia (abnormal hemoglobins). However, the phenotypic expression of hyperunstable hemoglobin variants often includes features of thalassemia, along with variable peripheral hemolysis. Hemoglobinopathies caused by highly unstable beta-chain variants have a dominant thalassemia-like phenotype, in which carriers have a clinical expression of thalassemia intermedia, but highly unstable alpha-globin variants are usually only phenotypically apparent when they interact with other alpha-thalassemia mutations. In a child with clinical and hematological features consistent with beta-thalassemia intermedia, DNA analysis excluded any beta-globin gene mutations but characterized a novel deletion cd37(C2)Pro>0 (Hb Heraklion) in the alpha1 globin gene, in trans to a common Mediterranean nondeletion alpha-thalassemia mutation (alpha(Hph)alpha). The deletion of proline at alpha37(C2) is predicted to result in severe instability of the variant hemoglobin, which on interaction with a synthesis-deficient alpha-thalassemia mutation causes a relatively severe dyserythropoietic anemia, representing an alternative phenotype associated with highly unstable alpha-chain variants. Hb Heraklion is the fourth highly unstable alpha-globin variant that we have observed in patients from Greece and Albania. Two variants involve the alpha2-globin gene: Hb Agrinio (alpha29(B10)Leu>Pro) and Hb Adana (alpha59(E8)Gly>Asp), and two the alpha1-gene: Hb Aghia Sophia (alpha62(E11)Val>0) and (Hb Heraklion a37(C2)Pro>0). Each has been observed on interaction with a different alpha-thalassemia mutation and the phenotypes associated with these highly unstable alpha-variants are presented.     

Author Index to Volume 27

A 50-year-old Japanese female patient was found to have hemolytic anemia. Isoelectrofocusing of her hemolysate revealed two abnormal hemoglobin bands, one of which was very close to the Hb A₂ band, and the other between the Hb A₂ and Hb F bands. CM-cellulose column chromatography of the globin prepared from the abnormal hemoglobin showed that the abnormal chain eluted faster than the normal β and δ chains; the β^X chain, however, did not separate from the normal β chain in urea cellulose acetate electrophoresis. An instability test of the patient's hemolysate revealed the presence of an unstable component. Structural analysis of the abnormal B chain indicated that the histidine residue at β92(F8) was replaced by an asparagine or aspartic acid residue. DNA amplified by poly-merase chain reaction was sequenced by the dideoxy method. The nucleotide sequence of the β92 codon was AAC instead of CAC, suggesting that the amino acid substitution corresponded to His→tAsn, which is the same as is found in Hb Redondo or ～92(F8)His→Asn→Asp.     

Hb Bronte or alpha93(FG5)Val-->Gly: a new unstable variant of the alpha2-globin gene,associated with a mild alpha(+)-thalassemia phenotype

We report a new unstable variant identified in three carriers of a family from East Sicily; it was named Hb Bronte after the place from which the family originated. DNA sequencing from nucleotides -181 to +894 (alpha1) and to +884 (alpha2) revealed a GTG-->GGG substitution at codon 93 of the alpha2-globin gene. The MCV and MCH values were at the lower end of the normal range in the carriers. On cation exchange high performance liquid chromatography (HPLC), the Hb A2 level was apparently increased to around 6%, and a small abnormal peak (0.3-0.4%) was detected after Hb A2. Two abnormal bands were detected by cellulose acetate electrophoresis: a major band (about 3-4%) migrated between Hb A and Hb F; a minor band (<1%) migrated between Hb A2 and carbonic anhydrase. Normal values of Hb A2 were detected by DEAE microchromatography. On reversed phase HPLC the variant chain was not detected, and most likely it was eluted with the alpha chain peak. The isopropanol stability test was very slightly positive in the carriers. Hemolytic symptoms were absent with the exception of indirect bilirubin, which was at high borderline in 2/3 carriers. In biosynthesis in vitro, the specific activity of the alpha chains was much higher than that of the beta-globin chains, and the alpha/beta biosynthetic ratio in the mother and proband was of the beta-thalassemia (thal) type (2.24 and 2.54, respectively). Time course experiments showed that the increase of the 3H-specific activity of the peak containing normal and variant alpha chains was not linear and was much higher than that of beta chains; moreover, the alpha/beta biosynthetic ratio varied during the 2 hours incubation.     

Multicenter validation of fully automated capillary electrophoresis method for diagnosis of thalassemias and hemoglobinopathies in Thailand

Thalassemias and hemoglobinopathies are highly prevalent in Thailand and other Southeast Asian countries. Accurate and precise separation of hemoglobin types, together with reliable quantitation, are essential for differential diagnosis of these diseases. Presented in this study is a multicenter validation of a fully automated capillary electrophoresis (CE) method for hemoglobin separation and quantitation involving four reference laboratories in Thailand. Analytical performance characteristics, including precision and accuracy were compared with existing validated HPLC and LPLC methods using 412 blood samples from unrelated subjects. Coefficient of variance of Hb A2 quantitation was 1.80-2.86, 1.26-5.13 and 1.08-6.66% for within run, between run and interlaboratory comparison, respectively. Results of Hb A2 and Hb F quantitated by the CE method correlates well with those of the two comparative methods (r = 0.98-0.99). The CE method correctly determined the genotypes (thalassemias and hemoglobin variants) of all blood samples tested. The major advantage of the CE system is its ability to separate and quantitate Hb A2, Hb E, Hb F, Hb H and Hb Bart's, which are important parameters required for diagnosis of thalassemias and hemoglobinopathies.