*4.1.2.4 Hemoglobin study by cation-exchange high performance liquid chromatography (HPLC)*

Cation-exchange HPLC has become the reference method for typing and quantitating hemoglobins in blood samples [19, 67, 68]. In this system, hemoglobins are dissolved in buffer having a pH of 6.4 that is less than pI of hemoglobins (6.5–7.5) and molecular net charge then is converted to be positive. Different hemoglobins then have different amount of positive charge which determines binding strength of hemoglobins to negatively charged resin. Hb Bart's has the weakest binding affinity, while Hb Constant Spring has the strongest binding affinity. Therefore, on passing external cation, the order of hemoglobins that are eluted fast to slowly should be as follows: Hb Bart's-HbH-HbF-HbAo-HbA2/E-Hb Constant Spring (**Figure 8**).

#### **Figure 8.**

*Hemoglobin pattern of cation-exchange HPLC of normal human adult: A2A. As shown in the figure, major hemoglobin is HbAo which accounts for 82.4%, while the minor HbA2 accounts for 2.6%. Other minor hemoglobins are labeled P2 and P3, which are Hbs A1a, A1b, and A1c mixture. HbF peak is negligible and reported as 0.0%. This HPLC result may be that of normal individuals or α-thalassemia carriers. It is noted that this kind of cationexchange HPLC pattern may be also observed in carriers of α-thalassemia 1 and carriers of α-thalassemia 2.*

**137**

**Figure 9.**

*and reported as 0.0%.*

*Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

reporting HbA2 as reported by the machine.

adults are A2A with HbA2 of 2.6 ± 0.38% [19].

platform.

HbA is normally derivatized to several fractions including the minor Hbs A1a, A1b, A1c, and major HbAo. The minor Hbs A1a, A1b, and A1c are eluted just after HbF. For routine Hb typing work, only the major HbAo is usually reported to clini-

HbE, which is common in Southeastern part of the world, is co-eluted with HbA2. However, most of the manufacturers design program to read hemoglobin peak at the A2 region as only HbA2. Therefore, the operator must be aware that if the percentage of A2-peak is more than 10, it is HbE plus HbA2 and indicates that the sample has HbE. The operator must report HbE or HbE plus HbA2, instead of

There are several manufacturers producing the HPLC machine in the world and the operating procedures as well as quality control protocols are established specifically for each brand. Most importantly, all of these brands generate identical separation peaks of hemoglobins. **Figure 8** shows example of hemoglobin pattern obtained from Variant™ Hemoglobin Analysis System, the widely used HPLC machine in Thailand. In this protocol, the hemoglobin types in normal human

Contrast to the CAE at pH 8.6, hemoglobins separated by the cation-exchange

*Hemoglobin pattern of cation-exchange HPLC of β-thalassemia carrier in human adult: A2A. As shown in the figure, major hemoglobin is HbAo which accounts for 78.6%, while the minor HbA2 accounts for 5.9%. Other minor hemoglobins are labeled P2 and P3, which are Hbs A1a, A1b, and A1c mixture. HbF peak is negligible* 

HPLC are automatically calculated for their proportions in blood. Therefore, both types and quantities of hemoglobins are usually obtained when run in this

cians. This makes sum of hemoglobin peaks does not equal to 100%.

#### *Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

*Beta Thalassemia*

β-thalassemia and β-hemoglobinopathies [6]. Patients with β-thalassemia disease who also inherit high HbF gene or quantitative trait loci (QTLs) will have mild clinical symptoms. Parents having high HbF gene can pass this gene to their β-thalassemia offspring. Thus, determining HbF in parents is useful in this way.

Cation-exchange HPLC has become the reference method for typing and quantitating hemoglobins in blood samples [19, 67, 68]. In this system, hemoglobins are dissolved in buffer having a pH of 6.4 that is less than pI of hemoglobins (6.5–7.5) and molecular net charge then is converted to be positive. Different hemoglobins then have different amount of positive charge which determines binding strength of hemoglobins to negatively charged resin. Hb Bart's has the weakest binding affinity, while Hb Constant Spring has the strongest binding affinity. Therefore, on passing external cation, the order of hemoglobins that are eluted fast to slowly should be as follows: Hb Bart's-HbH-HbF-HbAo-HbA2/E-Hb Constant Spring (**Figure 8**).

*Hemoglobin pattern of cation-exchange HPLC of normal human adult: A2A. As shown in the figure, major hemoglobin is HbAo which accounts for 82.4%, while the minor HbA2 accounts for 2.6%. Other minor hemoglobins are labeled P2 and P3, which are Hbs A1a, A1b, and A1c mixture. HbF peak is negligible and reported as 0.0%. This HPLC result may be that of normal individuals or α-thalassemia carriers. It is noted that this kind of cationexchange HPLC pattern may be also observed in carriers of α-thalassemia 1 and carriers of α-thalassemia 2.*

*4.1.2.4 Hemoglobin study by cation-exchange high performance liquid* 

*chromatography (HPLC)*

**136**

**Figure 8.**

HbA is normally derivatized to several fractions including the minor Hbs A1a, A1b, A1c, and major HbAo. The minor Hbs A1a, A1b, and A1c are eluted just after HbF. For routine Hb typing work, only the major HbAo is usually reported to clinicians. This makes sum of hemoglobin peaks does not equal to 100%.

HbE, which is common in Southeastern part of the world, is co-eluted with HbA2. However, most of the manufacturers design program to read hemoglobin peak at the A2 region as only HbA2. Therefore, the operator must be aware that if the percentage of A2-peak is more than 10, it is HbE plus HbA2 and indicates that the sample has HbE. The operator must report HbE or HbE plus HbA2, instead of reporting HbA2 as reported by the machine.

There are several manufacturers producing the HPLC machine in the world and the operating procedures as well as quality control protocols are established specifically for each brand. Most importantly, all of these brands generate identical separation peaks of hemoglobins. **Figure 8** shows example of hemoglobin pattern obtained from Variant™ Hemoglobin Analysis System, the widely used HPLC machine in Thailand. In this protocol, the hemoglobin types in normal human adults are A2A with HbA2 of 2.6 ± 0.38% [19].

Contrast to the CAE at pH 8.6, hemoglobins separated by the cation-exchange HPLC are automatically calculated for their proportions in blood. Therefore, both types and quantities of hemoglobins are usually obtained when run in this platform.

#### **Figure 9.**

*Hemoglobin pattern of cation-exchange HPLC of β-thalassemia carrier in human adult: A2A. As shown in the figure, major hemoglobin is HbAo which accounts for 78.6%, while the minor HbA2 accounts for 5.9%. Other minor hemoglobins are labeled P2 and P3, which are Hbs A1a, A1b, and A1c mixture. HbF peak is negligible and reported as 0.0%.*

#### **Figure 10.**

*Hemoglobin pattern of cation-exchange HPLC of HbE carrier in human adults: EA. As shown in the figure, major hemoglobin is HbAo which accounts for 59.2%, while the minor HbE (plus A2) accounts for 24.9%. Other minor hemoglobins are labeled P2 and P3, which are Hbs A1a, A1b, and A1c mixture. HbF peak is negligible and reported as 0.0%.*

#### **4.2 Cation-exchange HPLC hemoglobin patterns of β-thalassemia carriers and HbE carriers in human adults**

Hemoglobin patterns obtained from the cation-exchange HPLC of β-thalassemia and HbE are totally different. In carrier state, β-thalassemia carriers in adult life have normal Hb types for adult which is A2A, but HbA2 levels is increased to the levels of 5.9 ± 1.35% (**Figure 9**) [19]. HbE carriers in adult life have abnormal Hb typing by the cation-exchange HPLC which is AE with HbE (plus A2) of 27 ± 3.93% [19], as shown in **Figure 10**.

#### **4.3 Cation-exchange HPLC hemoglobin patterns of β-thalassemia diseases and HbE disease in human adults**

Hemoglobin patterns by the cation-exchange HPLC of adult β-thalassemia disease consist of several patterns depending on the combination of the abnormal β-thalassemia mutations.

#### *4.3.1 Homozygous β<sup>O</sup>***-***thalassemia*

Individuals of homozygous βO-thalassemia (βO/βO) are usually affected by the severe thalassemia disease and require regular blood transfusion. This group of patients

**139**

*4.3.2 Homozygous β<sup>+</sup>*

**Figure 12.**

*heterozygous β<sup>+</sup>*

**Figure 11.**

in this homozygous β<sup>+</sup>

*-thalassemia*

*Cation-exchange HPLC hemoglobin pattern A2FA seen in homozygous β<sup>+</sup>*

cation-exchange HPLC of the homozygous β<sup>+</sup>

and previously are classified as β-thalassemia intermedia. The β-thalassemia intermedia cases usually require no blood transfusion. Thus, now this group of patients is newly classified as non-transfusion dependent thalassemia (NTDT)

is quite resembling to that of transfused homozygous βO-thalassemia, that is, A2FA. However, single population of red blood cells on blood smear is also revealed

transfused homozygous βO-thalassemia. **Figure 12** shows hemoglobin pattern by

[69]. Hemoglobin pattern on cation-exchange HPLC of homozygous β<sup>+</sup>

*/βO-thalassemia (HbAo: 24.6%, HbF: 68.5%, HbA2: 3.9%).*


*-thalassemia and compound* 




Individuals of homozygous β<sup>+</sup>

*Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

is previously classified as thalassemia major, but now as transfusion dependent thalassemia [69]. Hence, hemoglobin patterns of homozygous βO-thalassemia in adult life of human should consist of HbF and HbA2 with no HbA prior to blood transfusion, that is, A2F (**Figure 11**). However, after recent blood transfusion, the hemoglobin types of

*Cation-exchange HPLC pattern of homozygous βO-thalassemia prior to blood transfusion, which is read as* 

*A2F (F: 97.8%, A2: 2.2%) (credit to Eaktong Limveeraprajak of Sawan Pracharak Hospital).*

A2FA are shown. HbA is certainly from the transfused blood (**Figure 12**).

*Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

is previously classified as thalassemia major, but now as transfusion dependent thalassemia [69]. Hence, hemoglobin patterns of homozygous βO-thalassemia in adult life of human should consist of HbF and HbA2 with no HbA prior to blood transfusion, that is, A2F (**Figure 11**). However, after recent blood transfusion, the hemoglobin types of A2FA are shown. HbA is certainly from the transfused blood (**Figure 12**).

#### **Figure 11.**

*Beta Thalassemia*

**138**

**4.2 Cation-exchange HPLC hemoglobin patterns of β-thalassemia carriers and** 

*Hemoglobin pattern of cation-exchange HPLC of HbE carrier in human adults: EA. As shown in the figure, major hemoglobin is HbAo which accounts for 59.2%, while the minor HbE (plus A2) accounts for 24.9%. Other minor hemoglobins are labeled P2 and P3, which are Hbs A1a, A1b, and A1c mixture. HbF peak is* 

and HbE are totally different. In carrier state, β-thalassemia carriers in adult life have normal Hb types for adult which is A2A, but HbA2 levels is increased to the levels of 5.9 ± 1.35% (**Figure 9**) [19]. HbE carriers in adult life have abnormal Hb typing by the cation-exchange HPLC which is AE with HbE (plus A2) of 27 ± 3.93%

**4.3 Cation-exchange HPLC hemoglobin patterns of β-thalassemia diseases and** 

Hemoglobin patterns by the cation-exchange HPLC of adult β-thalassemia disease consist of several patterns depending on the combination of the abnormal

Individuals of homozygous βO-thalassemia (βO/βO) are usually affected by the severe thalassemia disease and require regular blood transfusion. This group of patients

Hemoglobin patterns obtained from the cation-exchange HPLC of β-thalassemia

**HbE carriers in human adults**

**HbE disease in human adults**

[19], as shown in **Figure 10**.

*negligible and reported as 0.0%.*

**Figure 10.**

β-thalassemia mutations.

*4.3.1 Homozygous β<sup>O</sup>***-***thalassemia*

*Cation-exchange HPLC pattern of homozygous βO-thalassemia prior to blood transfusion, which is read as A2F (F: 97.8%, A2: 2.2%) (credit to Eaktong Limveeraprajak of Sawan Pracharak Hospital).*

#### **Figure 12.**

*Cation-exchange HPLC hemoglobin pattern A2FA seen in homozygous β<sup>+</sup> -thalassemia and compound heterozygous β<sup>+</sup> /βO-thalassemia (HbAo: 24.6%, HbF: 68.5%, HbA2: 3.9%).*

#### *4.3.2 Homozygous β<sup>+</sup> -thalassemia*

Individuals of homozygous β<sup>+</sup> -thalassemia always have mild clinical symptoms and previously are classified as β-thalassemia intermedia. The β-thalassemia intermedia cases usually require no blood transfusion. Thus, now this group of patients is newly classified as non-transfusion dependent thalassemia (NTDT) [69]. Hemoglobin pattern on cation-exchange HPLC of homozygous β<sup>+</sup> -thalassemia is quite resembling to that of transfused homozygous βO-thalassemia, that is, A2FA. However, single population of red blood cells on blood smear is also revealed in this homozygous β<sup>+</sup> -thalassemia, in contrast for dimorphic population in case of transfused homozygous βO-thalassemia. **Figure 12** shows hemoglobin pattern by cation-exchange HPLC of the homozygous β<sup>+</sup> -thalassemia.

#### *4.3.3 Compound heterozygous βO/β<sup>+</sup> -thalassemia*

Patients with compound heterozygous βO/β<sup>+</sup> -thalassemia always have severe disease and may require blood transfusion. Thus, they are classified as TDT. Hemoglobin patterns by cation-exchange HPLC of transfused and nontransfused cases is A2FA, being similar to homozygous β<sup>+</sup> -thalassemia (**Figure 12**). However, the compound heterozygous βO/β<sup>+</sup> -thalassemia has thalassemic red blood cell morphology like homozygous βO-thalassemia. In contrast, red blood cell morphology of homozygous β<sup>+</sup> -thalassemia is less abnormal than the other two β-thalassemia mentioned above.

Hb patterns by the cation-exchange HPLC of adult HbE disease also comprise several varieties depending on combination of β<sup>E</sup> mutation.

#### *4.3.4 Homozygous HbE*

Homozygous HbE (β<sup>E</sup> /β<sup>E</sup> ) is the mild form of β-thalassemia disease. The patients usually have good clinical symptom with only mild anemia with no need of blood transfusion. Thus, cation-exchange HPLC always shows HbE as major hemoglobin and HbF as the minor hemoglobin; that is, EF (**Figure 13**). Sometime, this hemoglobin type of EF may be confused with that of HbE/βO-thalassemia as HbF in some cases of the later condition may be as low as 4.5% [70] and 2.1% [19]. This low level of HbF may overlap with that seen in homozygous HbE (4.3 ± 2.66%) [19]. Again, red blood cell morphology will help identify if the case is homozygous HbE or the HbE/βO-thalassemia. Red blood cell morphology on

#### **Figure 13.**

*Hemoglobin pattern by cation-exchange HPLC of homozygous HbE in adults. The major peak contains HbE plus HbA2, but it is labeled A2 by software. Thus, level of HbE plus HbA2 in this case is 77.3, and that of HbF is 1.7%. This case has no HbA, but the software mislabeled the HbA1 fraction as Ao.*

**141**

the chromatogram.

*Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

Individuals with compound heterozygote of β<sup>E</sup>

βO-thalassemia.

*4.3.5 Compound heterozygous β<sup>E</sup>*

recent blood transfusion (**Figure 14**).

*4.3.6 Compound heterozygous β<sup>E</sup>*

**Figure 14.**

blood smear stained with Wright-Giemsa stain is totally different between homozygous HbE and HbE/βO-thalassemia. In homozygous HbE, mild change of red blood cell morphology with considerable amount of target cells is usually observed. In contrast, thalassemia type of red blood cell morphology is typical for the HbE/

 *and βO-thalassemia*

affected by the thalassemia disease and some require blood transfusion. Therefore,

(61.2 ± 13.6% HbE, 31.1 ± 14.5% HbF) prior to blood transfusion [19] and EFA after

the hemoglobin patterns by cation-exchange HPLC of this case will be EF

 *and β<sup>+</sup>*

should be EFA. This is because some β-globin chains are still produced.

Individuals with compound heterozygote of β<sup>E</sup>

*4.3.7 Double form of HbE carrier and HbH disease*

HPLC (**Figure 15**). This is why it is called AEBart's disease.

*4.3.8 Double form of HbE homozygote and HbH disease*

*-thalassemia*

clinical symptoms and classified as NTDT. Therefore, interference of transfused blood is not possible. The cation-exchange HPLC pattern of hemoglobin in this case

*Hemoglobin pattern in cation-exchange HPLC of cases with HbE/βO-thalassemia (F 53.1%, E 35.8%).*

This thalassemia syndrome is conventionally termed AEBart's disease. This is a mild form of α-thalassemia syndrome, and blood transfusion is not required. Thus, transfused blood would not also interfere result reading in this situation. Hemoglobins A, E (with A2), Bart's are always seen under the cation-exchange

This is also a mild form of α-thalassemia disease that shows hemoglobins E, F, and Bart's in the cation-exchange HPLC. It is thus called EFBart's disease. By running the cation-exchange HPLC, Hbs E, F, and Bart's are always seen in

and β<sup>+</sup>


and βO-thalassemia are always

*Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

*Beta Thalassemia*

*4.3.3 Compound heterozygous βO/β<sup>+</sup>*

morphology of homozygous β<sup>+</sup>

*4.3.4 Homozygous HbE*

Homozygous HbE (β<sup>E</sup>

β-thalassemia mentioned above.

Patients with compound heterozygous βO/β<sup>+</sup>

However, the compound heterozygous βO/β<sup>+</sup>

several varieties depending on combination of β<sup>E</sup>

/β<sup>E</sup>

transfused cases is A2FA, being similar to homozygous β<sup>+</sup>

*-thalassemia*

severe disease and may require blood transfusion. Thus, they are classified as TDT. Hemoglobin patterns by cation-exchange HPLC of transfused and non-

blood cell morphology like homozygous βO-thalassemia. In contrast, red blood cell

Hb patterns by the cation-exchange HPLC of adult HbE disease also comprise

patients usually have good clinical symptom with only mild anemia with no need of blood transfusion. Thus, cation-exchange HPLC always shows HbE as major hemoglobin and HbF as the minor hemoglobin; that is, EF (**Figure 13**). Sometime, this hemoglobin type of EF may be confused with that of HbE/βO-thalassemia as HbF in some cases of the later condition may be as low as 4.5% [70] and 2.1% [19]. This low level of HbF may overlap with that seen in homozygous HbE (4.3 ± 2.66%) [19]. Again, red blood cell morphology will help identify if the case is homozygous HbE or the HbE/βO-thalassemia. Red blood cell morphology on

*Hemoglobin pattern by cation-exchange HPLC of homozygous HbE in adults. The major peak contains HbE plus HbA2, but it is labeled A2 by software. Thus, level of HbE plus HbA2 in this case is 77.3, and that of HbF is* 

*1.7%. This case has no HbA, but the software mislabeled the HbA1 fraction as Ao.*




mutation.

) is the mild form of β-thalassemia disease. The


**140**

**Figure 13.**

blood smear stained with Wright-Giemsa stain is totally different between homozygous HbE and HbE/βO-thalassemia. In homozygous HbE, mild change of red blood cell morphology with considerable amount of target cells is usually observed. In contrast, thalassemia type of red blood cell morphology is typical for the HbE/ βO-thalassemia.

#### *4.3.5 Compound heterozygous β<sup>E</sup> and βO-thalassemia*

Individuals with compound heterozygote of β<sup>E</sup> and βO-thalassemia are always affected by the thalassemia disease and some require blood transfusion. Therefore, the hemoglobin patterns by cation-exchange HPLC of this case will be EF (61.2 ± 13.6% HbE, 31.1 ± 14.5% HbF) prior to blood transfusion [19] and EFA after recent blood transfusion (**Figure 14**).

**Figure 14.** *Hemoglobin pattern in cation-exchange HPLC of cases with HbE/βO-thalassemia (F 53.1%, E 35.8%).*

#### *4.3.6 Compound heterozygous β<sup>E</sup> and β<sup>+</sup> -thalassemia*

Individuals with compound heterozygote of β<sup>E</sup> and β<sup>+</sup> -thalassemia have mild clinical symptoms and classified as NTDT. Therefore, interference of transfused blood is not possible. The cation-exchange HPLC pattern of hemoglobin in this case should be EFA. This is because some β-globin chains are still produced.

#### *4.3.7 Double form of HbE carrier and HbH disease*

This thalassemia syndrome is conventionally termed AEBart's disease. This is a mild form of α-thalassemia syndrome, and blood transfusion is not required. Thus, transfused blood would not also interfere result reading in this situation. Hemoglobins A, E (with A2), Bart's are always seen under the cation-exchange HPLC (**Figure 15**). This is why it is called AEBart's disease.

#### *4.3.8 Double form of HbE homozygote and HbH disease*

This is also a mild form of α-thalassemia disease that shows hemoglobins E, F, and Bart's in the cation-exchange HPLC. It is thus called EFBart's disease. By running the cation-exchange HPLC, Hbs E, F, and Bart's are always seen in the chromatogram.

#### **Figure 15.**

*Cation-exchange HPLC of hemoglobin component in AEBart's disease. HbE: 13.5%, HbA0: 71.8%, Hb Bart's (no numeric proportion as the analysis software was not designed for Hb Bart's quantification).*

#### *4.3.9 Double form of HbE/βO-thalassemia and HbH disease*

This is a rare form of thalassemia syndrome. On running in the cation-exchange HPLC, EFBart's pattern of hemoglobin is also seen, being similar to the double form of homozygous HbE and HbH disease. Red blood cell morphology on blood smear may help differentiate these two conditions, but skillful personnels are needed to examine red blood cell morphology. However, DNA analysis in the only technique that can correctly differentiate this EFBart's syndrome.

#### **5. Hemoglobin study by capillary zone electrophoresis (CZE)**

Capillary zone electrophoresis (CZE) has been introduced for use as a tool for analysis of hemoglobin variants [23–27, 71]. Conventionally, separation of hemoglobin is performed in alkaline condition, in which HbH has the maximum molecular negative charge, followed, respectively, by Hb Bart's, HbA, HbF, HbsA2/E, and Hb Constant Spring. The separation is based on high voltage (7500 V) and electro-endo-osmotic force (EOF). Hemoglobins are forced in the system to move from anode to cathode with the cuvette placed at the cathodic end. Once hemoglobin band moves into the cuvette, the 415-nm absorbance is measured and the light signals are converted by the software to electropherogram. Each hemoglobin has its own location or zone in the electropherogram, HbCS-zone 2: Z(C), HbA2-zone 3: Z(A2), HbE-zone 4: Z(E), HbF-zone 7: Z(F), HbA-zone 9: Z (A), Hb Bart's – zone 12, and Hb H – zone 15. HbE and HbA2 are clearly separated by this system (**Figures 16** and **17**).

#### **5.1 The CZE pattern of β-thalassemia carriers and HbE carriers**

In β-thalassemia carrier at adult life, the CZE pattern of hemoglobin is similar to that obtained from cation-exchange HPLC. The normal hemoglobin typing result of A2A or A2FA with HbA2 levels of more than 3.5% (5.4 ± 0.5%) and HbF levels of less than 2% (0.9 ± 1.4%) are always observed [27] (**Figure 18**).

In HbE carrier of adult life, the hemoglobin pattern of CZE is different from that of cation-exchange HPLC. HbE and HbA2 co-eluted in the cation-exchange HPLC. In CZE, HbE moves behind HbA2. Thus, hemoglobin pattern of HbE carrier

**143**

**Figure 16.**

**Figure 17.**

*Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

in CZE should be A2EFA. The percentage of these hemoglobins is as follows: HbA2: 3.5 ± 0.4%, HbE: 25.6 ± 1.4%, and HbF: 0.4 ± 0.8% [27]. HbE level in the CZE system is usually lower than that obtained from cation-exchange HPLC (27.8 ± 7.5%). This is due to the fact that the level of HbE from HPLC is the sum of HbE and HbA2 that are co-eluted, while only HbE is reported in the CZE system. Thus, performers must be careful in reporting HbE. HbA2 level is slightly elevated. This confirms that

*CZE electropherogram of normal human hemoglobins. HbA: 94.3%, HbF: 2.4%, HbA2: 3.3%.*

CZE pattern of hemoglobins in β-thalassemia disease in adults depends on types of the disease. Although, principles of separation are different, the patterns of hemoglobin

HbE carrier also acts as mild β-thalassemia carrier (**Figure 19**).

*CZE pattern of hemoglobins from zone (Z) 1 to zone (Z) 15.*

**5.2 The CZE pattern in β-thalassemia disease and HbE disease**

*Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

**Figure 16.**

*Beta Thalassemia*

**Figure 15.**

*4.3.9 Double form of HbE/βO-thalassemia and HbH disease*

that can correctly differentiate this EFBart's syndrome.

**5. Hemoglobin study by capillary zone electrophoresis (CZE)**

**5.1 The CZE pattern of β-thalassemia carriers and HbE carriers**

less than 2% (0.9 ± 1.4%) are always observed [27] (**Figure 18**).

This is a rare form of thalassemia syndrome. On running in the cation-exchange HPLC, EFBart's pattern of hemoglobin is also seen, being similar to the double form of homozygous HbE and HbH disease. Red blood cell morphology on blood smear may help differentiate these two conditions, but skillful personnels are needed to examine red blood cell morphology. However, DNA analysis in the only technique

*Cation-exchange HPLC of hemoglobin component in AEBart's disease. HbE: 13.5%, HbA0: 71.8%, Hb Bart's* 

*(no numeric proportion as the analysis software was not designed for Hb Bart's quantification).*

Capillary zone electrophoresis (CZE) has been introduced for use as a tool for analysis of hemoglobin variants [23–27, 71]. Conventionally, separation of hemoglobin is performed in alkaline condition, in which HbH has the maximum molecular negative charge, followed, respectively, by Hb Bart's, HbA, HbF, HbsA2/E, and Hb Constant Spring. The separation is based on high voltage (7500 V) and electro-endo-osmotic force (EOF). Hemoglobins are forced in the system to move from anode to cathode with the cuvette placed at the cathodic end. Once hemoglobin band moves into the cuvette, the 415-nm absorbance is measured and the light signals are converted by the software to electropherogram. Each hemoglobin has its own location or zone in the electropherogram, HbCS-zone 2: Z(C), HbA2-zone 3: Z(A2), HbE-zone 4: Z(E), HbF-zone 7: Z(F), HbA-zone 9: Z (A), Hb Bart's – zone 12, and Hb H – zone 15. HbE and HbA2 are clearly separated by this system

In β-thalassemia carrier at adult life, the CZE pattern of hemoglobin is similar to that obtained from cation-exchange HPLC. The normal hemoglobin typing result of A2A or A2FA with HbA2 levels of more than 3.5% (5.4 ± 0.5%) and HbF levels of

In HbE carrier of adult life, the hemoglobin pattern of CZE is different from that of cation-exchange HPLC. HbE and HbA2 co-eluted in the cation-exchange HPLC. In CZE, HbE moves behind HbA2. Thus, hemoglobin pattern of HbE carrier

**142**

(**Figures 16** and **17**).

*CZE electropherogram of normal human hemoglobins. HbA: 94.3%, HbF: 2.4%, HbA2: 3.3%.*

**Figure 17.** *CZE pattern of hemoglobins from zone (Z) 1 to zone (Z) 15.*

in CZE should be A2EFA. The percentage of these hemoglobins is as follows: HbA2: 3.5 ± 0.4%, HbE: 25.6 ± 1.4%, and HbF: 0.4 ± 0.8% [27]. HbE level in the CZE system is usually lower than that obtained from cation-exchange HPLC (27.8 ± 7.5%). This is due to the fact that the level of HbE from HPLC is the sum of HbE and HbA2 that are co-eluted, while only HbE is reported in the CZE system. Thus, performers must be careful in reporting HbE. HbA2 level is slightly elevated. This confirms that HbE carrier also acts as mild β-thalassemia carrier (**Figure 19**).

#### **5.2 The CZE pattern in β-thalassemia disease and HbE disease**

CZE pattern of hemoglobins in β-thalassemia disease in adults depends on types of the disease. Although, principles of separation are different, the patterns of hemoglobin

**Figure 18.**

*CZE pattern of hemoglobins of β-thalassemia carrier.*

**145**

heterozygous β<sup>+</sup>

**Figure 20.**

*36.4% HbF.*

each hemoglobin (**Figure 20**).

α-thalassemia carriers [32, 75].

*Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

in β-thalassemia disease and HbE disease obtained from CZE are quite similar to those obtained from cation-exchange HPLC. For homozygous βO-thalassemia, A2F is the

*CZE pattern of hemoglobins of HbE/βO-thalassemia, composing of A2EF with 4.7% HbA2, 58.9% HbE, and* 

Thalassemia and hemoglobinopathies can be identified accurately by using monoclonal antibodies (mAbs) against human hemoglobins [72, 73]. Application of mAb-based protocols aims primarily to identify the carriers of thalassemia and hemoglobinopathies. For instance, α-thalassemia carrier can be detected by using mAbs against Hb Bart's [29] and HbH [74]. Immunochromatographic strip test utilizing mAb to Hb Bart's was produced and successfully applied for screening

/βO-thalassemia, the A2FA is generally seen in CZE platform. CZE patterns of hemoglobins in HbE disease in adults also depend on types of the diseases. However, as HbE and HbA2 are clearly separated in the CZE platform, these two hemoglobins must be separately reported. For example, in HbE/βOthalassemia, for example, A2EF must be reported together with the proportion of


typical hemoglobin typing results. For homozygous β<sup>+</sup>

**6. Hemoglobin study by monoclonal antibody**

**Figure 19.** *CZE pattern of hemoglobin of HbE carrier.*


#### **Figure 20.**

*Beta Thalassemia*

**144**

**Figure 19.**

**Figure 18.**

*CZE pattern of hemoglobins of β-thalassemia carrier.*

*CZE pattern of hemoglobin of HbE carrier.*

*CZE pattern of hemoglobins of HbE/βO-thalassemia, composing of A2EF with 4.7% HbA2, 58.9% HbE, and 36.4% HbF.*

in β-thalassemia disease and HbE disease obtained from CZE are quite similar to those obtained from cation-exchange HPLC. For homozygous βO-thalassemia, A2F is the typical hemoglobin typing results. For homozygous β<sup>+</sup> -thalassemia and compound heterozygous β<sup>+</sup> /βO-thalassemia, the A2FA is generally seen in CZE platform.

CZE patterns of hemoglobins in HbE disease in adults also depend on types of the diseases. However, as HbE and HbA2 are clearly separated in the CZE platform, these two hemoglobins must be separately reported. For example, in HbE/βOthalassemia, for example, A2EF must be reported together with the proportion of each hemoglobin (**Figure 20**).

#### **6. Hemoglobin study by monoclonal antibody**

Thalassemia and hemoglobinopathies can be identified accurately by using monoclonal antibodies (mAbs) against human hemoglobins [72, 73]. Application of mAb-based protocols aims primarily to identify the carriers of thalassemia and hemoglobinopathies. For instance, α-thalassemia carrier can be detected by using mAbs against Hb Bart's [29] and HbH [74]. Immunochromatographic strip test utilizing mAb to Hb Bart's was produced and successfully applied for screening α-thalassemia carriers [32, 75].

#### **Figure 21.**

*HbA2 levels determined by sandwich ELISA set up in the author's laboratory. Note that HbA2 levels in β-thalassemia trait are higher than those in normal, HbE trait, homozygous HbE, and suspected α-thalassemia trait (modified from [30]).*

The β-thalassemia carrier can also be identified by using antigen-antibody reaction. Since elevated HbA2 level has been shown to be diagnostic marker of the β-thalassemia carrier, mAbs against δ-globin chain of HbA2 were produced and ELISA set up to quantify HbA2 levels by Shyamala et al. [76]. Using this ELISA, Shyamala found mean value of HbA2 in normal and β-thalassemia carrier to be 2.5 and 5.4%, respectively. The mAb against HbA2 was also produced and sandwich ELISA developed in the author's laboratory [30]. Under this developed sandwich ELISA, Kuntaruk found that the levels of HbA2 between normal and β-thalassemia carrier were also significantly different (**Figure 21**). Thus, the β-thalassemia carrier can be identified by the sandwich ELISA to quantify HbA2 level.

### **7. DNA analysis for β-thalassemia and HbE**

Analysis of mutations in β-globin gene to identify β-thalassemia and HbE is now performed routinely in most laboratories. The finding of the causative point mutations in the β-globin gene provides definite diagnosis of these disorders. More than 900 point mutations have been reported for β-thalassemia and β-hemoglobinopathies (Globin Gene Server: http://globin.cse.psu.edu/). Certain ethic groups have their own pattern of point mutations of β-globin gene [2, 4, 77, 78].

There are several allele-specific PCR protocols for detecting both carrier and disease state of β-thalassemia and HbE. These include mutagenically separated (MS)-PCR [35] and amplification refractory mutation system (ARMS)-PCR [79]. These two protocols were modified and adapted in author's laboratory. Another allele-specific PCR protocol was established in the author's laboratory and named "Multiplex Allele-Specific (MAS)-PCR" [29].

#### **7.1 Identification of β-thalassemia and HbE by MS-PCR**

The MS-PCR was used to detect β-globin gene mutations by several centers. In author's laboratory, this PCR protocol was modified and adapted to identify

**147**

**Figure 22.**

*amplified products are seen.*

β41/42/β<sup>T</sup>

primer" for β

*Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

the protocol described previously.

*7.1.1 MS-PCR for β 41/42 mutation*

at codon 17 (β

the β-globin gene mutations commonly found in Thai individuals. These included TTCT deletion or 4 bp-deletion at codons 41/42 (β41/42) and A > T substitution

account for approximately 67.5% in Thais by author's survey [29] and 83.9% by others' studies [81]. The MS-PCR was performed in the author's laboratory under

**Procedure**: The 25-μL PCR is performed containing 250 ng genomic DNA, 200 μM of each dNTP; 0.5 units Taq DNA polymerase, 100 ng of "Common primer" for β41/42; 5′-TCA TTC GTC TGT CCA TTC TAA AC-3′, 150 ng of "Normal primer" for β41/42; 5′-TTC CCA CCA TTA GGC TGC TGG TGG TCT ACC CTT GGA CCC AGA GGT TCT T-3′, 250 ng of "Mutant primer" for β41/42; 5′-ACC CTT GGA CCC AGA GGT TGA G-3′, 10 mM Tris pH 8.8; 50 mM KCl and 1.5 mM of MgCl2. **Thermal cycles**: A total of 35 thermal cycles was carried out with each cycle comprising DNA denaturation at 94°C for 1 min, primer annealing at 56°C for 1 min, and primer extension at 72°C for 1 min; the initial denaturation was extended to 4 min while the final extension was prolonged to 5 min.

**Detection of amplified products**: The amplified products were separated in 2.5% agarose gel electrophoresis at 120 V for 15–20 min before visualizing with a UV-transilluminator. The fragments sizing 324 and 351 bp indicate presence and

**Interpretation**: Samples having only 324-bp amplified fragments are homozygote for β41/42 with genotype β41/42/β41/42. Samples having only 351-bp fragments are

(A represents HbA; T represents other types of β-globin gene mutation).

17; 5′-GGC AGA GAG AGT CAG TGC CTA-3′, 150 ng of "Normal

**Procedure**: The 25-μL PCR is performed containing 250 ng genomic DNA, 200 μM of each dNTP; 0.5 units Taq DNA polymerase, 100 ng of "Common

*MS-PCR for detecting β41/42 mutation. Lanes 1 and 3 are negative for the β41/42 mutations as only 351-bp amplified products are seen. Lanes 2, 4, and 5 are heterozygote for the β41/42 mutation as both 3510 and 324-bp* 

other types of β-globin gene mutation). Samples having both 324 and 351-bp amplified products are heterozygote for the β41/42 with genotype of either β41/42/βA or

/β<sup>T</sup>

(A represents HbA; T represents

absence of the β41/42 mutation, respectively (**Figure 22**).

negative for the β41/42 with genotype βA/βA or β<sup>T</sup>

*7.1.2 MS-PCR for β17 mutation*

17) of β-globin gene [80]. These two mutations have been shown to

*Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

*Beta Thalassemia*

**Figure 21.**

*trait (modified from [30]).*

The β-thalassemia carrier can also be identified by using antigen-antibody reaction. Since elevated HbA2 level has been shown to be diagnostic marker of the β-thalassemia carrier, mAbs against δ-globin chain of HbA2 were produced and ELISA set up to quantify HbA2 levels by Shyamala et al. [76]. Using this ELISA, Shyamala found mean value of HbA2 in normal and β-thalassemia carrier to be 2.5 and 5.4%, respectively. The mAb against HbA2 was also produced and sandwich ELISA developed in the author's laboratory [30]. Under this developed sandwich ELISA, Kuntaruk found that the levels of HbA2 between normal and β-thalassemia carrier were also significantly different (**Figure 21**). Thus, the β-thalassemia car-

*HbA2 levels determined by sandwich ELISA set up in the author's laboratory. Note that HbA2 levels in β-thalassemia trait are higher than those in normal, HbE trait, homozygous HbE, and suspected α-thalassemia* 

rier can be identified by the sandwich ELISA to quantify HbA2 level.

Analysis of mutations in β-globin gene to identify β-thalassemia and HbE is now performed routinely in most laboratories. The finding of the causative point mutations in the β-globin gene provides definite diagnosis of these disorders. More than 900 point mutations have been reported for β-thalassemia and β-hemoglobinopathies (Globin Gene Server: http://globin.cse.psu.edu/). Certain ethic groups have their own pattern of point mutations of β-globin gene [2, 4, 77, 78]. There are several allele-specific PCR protocols for detecting both carrier and disease state of β-thalassemia and HbE. These include mutagenically separated (MS)-PCR [35] and amplification refractory mutation system (ARMS)-PCR [79]. These two protocols were modified and adapted in author's laboratory. Another allele-specific PCR protocol was established in the author's laboratory and named

The MS-PCR was used to detect β-globin gene mutations by several centers. In author's laboratory, this PCR protocol was modified and adapted to identify

**7. DNA analysis for β-thalassemia and HbE**

"Multiplex Allele-Specific (MAS)-PCR" [29].

**7.1 Identification of β-thalassemia and HbE by MS-PCR**

**146**

the β-globin gene mutations commonly found in Thai individuals. These included TTCT deletion or 4 bp-deletion at codons 41/42 (β41/42) and A > T substitution at codon 17 (β 17) of β-globin gene [80]. These two mutations have been shown to account for approximately 67.5% in Thais by author's survey [29] and 83.9% by others' studies [81]. The MS-PCR was performed in the author's laboratory under the protocol described previously.

#### *7.1.1 MS-PCR for β 41/42 mutation*

**Procedure**: The 25-μL PCR is performed containing 250 ng genomic DNA, 200 μM of each dNTP; 0.5 units Taq DNA polymerase, 100 ng of "Common primer" for β41/42; 5′-TCA TTC GTC TGT CCA TTC TAA AC-3′, 150 ng of "Normal primer" for β41/42; 5′-TTC CCA CCA TTA GGC TGC TGG TGG TCT ACC CTT GGA CCC AGA GGT TCT T-3′, 250 ng of "Mutant primer" for β41/42; 5′-ACC CTT GGA CCC AGA GGT TGA G-3′, 10 mM Tris pH 8.8; 50 mM KCl and 1.5 mM of MgCl2.

**Thermal cycles**: A total of 35 thermal cycles was carried out with each cycle comprising DNA denaturation at 94°C for 1 min, primer annealing at 56°C for 1 min, and primer extension at 72°C for 1 min; the initial denaturation was extended to 4 min while the final extension was prolonged to 5 min.

**Detection of amplified products**: The amplified products were separated in 2.5% agarose gel electrophoresis at 120 V for 15–20 min before visualizing with a UV-transilluminator. The fragments sizing 324 and 351 bp indicate presence and absence of the β41/42 mutation, respectively (**Figure 22**).

**Interpretation**: Samples having only 324-bp amplified fragments are homozygote for β41/42 with genotype β41/42/β41/42. Samples having only 351-bp fragments are negative for the β41/42 with genotype βA/βA or β<sup>T</sup> /β<sup>T</sup> (A represents HbA; T represents other types of β-globin gene mutation). Samples having both 324 and 351-bp amplified products are heterozygote for the β41/42 with genotype of either β41/42/βA or β41/42/β<sup>T</sup> (A represents HbA; T represents other types of β-globin gene mutation).

#### *7.1.2 MS-PCR for β17 mutation*

**Procedure**: The 25-μL PCR is performed containing 250 ng genomic DNA, 200 μM of each dNTP; 0.5 units Taq DNA polymerase, 100 ng of "Common primer" for β 17; 5′-GGC AGA GAG AGT CAG TGC CTA-3′, 150 ng of "Normal

#### **Figure 22.**

*MS-PCR for detecting β41/42 mutation. Lanes 1 and 3 are negative for the β41/42 mutations as only 351-bp amplified products are seen. Lanes 2, 4, and 5 are heterozygote for the β41/42 mutation as both 3510 and 324-bp amplified products are seen.*

#### **Figure 23.**

*MS-PCR for identifying β17 mutation. Lanes 4, 5, 6, and 8 are negative for the β17 mutation as only 190-bp amplified products are seen. Lanes 7 is homozygote for the β17 mutation as only 170-bp amplified products is seen. Lanes 1, 2, and 3 are heterozygote for the β17 mutation since both 170 and 190-bp amplified products are seen.*

primer" for β 17; 5′-ACC TGA CTC CTG AGG AGA AGA CTG CCG TTA CTG CCC TGT GGG ACA-3′, 100 ng of "Mutant primer" for β 17; 5′-TCT GCC GTT ACT GCC CTG TGG CAC-3′, 10 mM Tris pH 8.8; 50 mM KCl and 1.5 mM of MgCl2.

**Thermal cycles**: A total of 35 thermal cycles was carried out with each cycle comprising DNA denaturation at 94°C for 1 min, primer annealing at 59°C for 1 min, and primer extension at 72°C for 1 min; the initial denaturation was extended to 4 min while the final extension was prolonged to 5 min.

**Detection of amplified products**: The amplified products were separated in 2.5% agarose gel electrophoresis at 120 V for 15–20 min before visualizing with a UV-transilluminator. The fragments sizing 170 and 190 bp indicate presence and absence of the β 17 mutation, respectively (**Figure 23**).

**Interpretation**: Samples having only 170-bp amplified fragments are homozygote for β 17 with genotype β 17/β 17. Samples having only 190-bp fragments are negative for the β 17 with genotype βA/βA or β<sup>T</sup> /β<sup>T</sup> (A represents HbA; T represents other types of β-globin gene mutation). Samples having both 170 and 190-bp amplified products are heterozygote for the β 17 with genotype of either β 17/βA or β 17/β<sup>T</sup> (A represents HbA; T represents other types of β-globin gene mutation).

#### *7.1.3 MS-PCR for β<sup>E</sup> mutation or HbE*

**Procedure**: The 25-μL PCR is performed containing 250 ng genomic DNA, 200 μM of each dNTP; 0.5 units Taq DNA polymerase, 100 ng of "Common primer" for β<sup>E</sup> ; 5′-GGC AGA GAG AGT CAG TGC CTA-3′, 100 ng of "Normal primer" for βE ; 5′-CGT GGA TGA AGT TGG TGG AG-3′, 150 ng of "Mutant primer" for β<sup>E</sup> ; 5′-CTG CCC TGT GGG CAA GGT GAA CGT GGA TGA AGT TGG TGG AA-3′, 10 mM Tris pH 8.8; 50 mM KCl and 1.25 mM of MgCl2.

**Thermal cycles**: A total of 35 thermal cycles was carried out with each cycle comprising DNA denaturation at 94°C for 1 min, primer annealing at 56°C for 1 min and primer extension at 72°C for 1 min; the initial denaturation was extended to 4 min while the final extension was prolonged to 5 min.

**Detection of amplified products**: The amplified products were separated in 2.5% agarose gel electrophoresis at 120 V for 15–20 min before visualizing with a UV-transilluminator. The fragments sizing 160 and 138 bp indicate presence and absence of the β<sup>E</sup> mutation, respectively (**Figure 24**).

**Interpretation**: Samples having only 160-bp amplified fragments are homozygote for β<sup>E</sup> with genotype β<sup>E</sup> /β<sup>E</sup> . Samples having only 138-bp fragments are negative for the β<sup>E</sup> with genotype βA/βA or β<sup>T</sup> /β<sup>T</sup> (A represents HbA; T represents other types

**149**

*Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

ucts are heterozygote for the β<sup>E</sup>

**Figure 24.**

*MS-PCR for identifying β<sup>E</sup>*

internal control for the ARMS-PCR.

**8.1 ARMS-PCR for β41/42**

of β-globin gene mutation). Samples having both 138 and 160-bp amplified prod-

*is seen. Lane 2 is heterozygote for βE mutation as both 138-bp and 160-bp amplified products are seen.*

HbA; T represents other types of β-globin gene mutation).

**8. Identification of β-thalassemia and HbE by ARMS-PCR**

with genotype of either β<sup>E</sup>

 *mutation. Lane 1 is negative for the βE mutation as only 138-bp amplified products* 

ARMS-PCR was established by Old et al. [79]. This technique also uses three oligonucleotide primers. However, the length of normal and mutant primers is similar. Therefore, size of normal and mutant amplified products is the same and cannot be separated in the agarose gel electrophoresis. Thus, two PCRs must be performed in the ARMS-PCR. Both PCRs have the same ingredients, except normal and mutant oligonucleotide primers are added in separated reaction tubes (M and N-tube). In addition, a pair of oligonucleotide primers specific to other gene must also be added into both PCRs. The amplified products obtained by this pair of primers are the

**Procedure**: Two 25-μL reactions are performed; M-reaction and N-reaction. Both M and N-reactions contain 150 ng genomic DNA, 200 μM of each dNTP; 0.6 units Taq DNA polymerase, 0.2 μM of "S-primer"; 5′-ACC TCA CCC TGT GGA GCC AC-3′, 0.15 μM of "M41/42 primer"; 5′-GAG TGG ACA GAT CCC CAA AGG ACT CAA CCT−3′ (for M-reaction only), 0.15 μM of "N41/42 primer"; 5′-GAG TGG ACA GAT CCC CAA AGG ACT CAA AGA-3′ (for N-reaction only), 0.2 μM of "P1 primer"; 5′-GCG ATC TGG GCT CTG TGT TCT-3′, 0.2 μM of "P2 primer"; 5′-GTT CCC TGA

GCC CCG ACA CG-3′, 10 mM Tris pH 8.8; 50 mM KCl and 1.5 mM of MgCl2.

5 min while the final extension was prolonged to 5 min.

**Thermal cycles**: A total of 35 thermal cycles was carried out with each cycle comprising DNA denaturation at 95°C for 1 min, primer annealing at 65°C for 1 min and primer extension at 72°C for 1 min; the initial denaturation was extended to

**Detection of amplified products**: The amplified products were separated in 2.5% agarose gel electrophoresis at 120 V for 15–20 min before visualizing with a UV-transilluminator. The fragments sizing 439 bp is the specific amplified products,

and the PCR products sizing 314 bp are the control products (**Figure 25**).

/βA or β<sup>E</sup>

/β<sup>T</sup>

(A represents

*Laboratory Diagnosis of β-Thalassemia and HbE DOI: http://dx.doi.org/10.5772/intechopen.90317*

**Figure 24.**

*Beta Thalassemia*

primer" for β

**Figure 23.**

absence of the β

*7.1.3 MS-PCR for β<sup>E</sup>*

absence of the β<sup>E</sup>

with genotype β<sup>E</sup>

with genotype βA/βA or β<sup>T</sup>

gote for β<sup>E</sup>

for the β<sup>E</sup>

17 with genotype β

products are heterozygote for the β

gote for β

for β<sup>E</sup>

βE

tive for the β

TGT GGG ACA-3′, 100 ng of "Mutant primer" for β

CTG TGG CAC-3′, 10 mM Tris pH 8.8; 50 mM KCl and 1.5 mM of MgCl2.

17 mutation, respectively (**Figure 23**).

represents HbA; T represents other types of β-globin gene mutation).

17/β

 *mutation or HbE*

10 mM Tris pH 8.8; 50 mM KCl and 1.25 mM of MgCl2.

4 min while the final extension was prolonged to 5 min.

/β<sup>E</sup>

17 with genotype βA/βA or β<sup>T</sup>

**Thermal cycles**: A total of 35 thermal cycles was carried out with each cycle comprising DNA denaturation at 94°C for 1 min, primer annealing at 59°C for 1 min, and primer extension at 72°C for 1 min; the initial denaturation was extended to 4 min while the final extension was prolonged to 5 min.

*MS-PCR for identifying β17 mutation. Lanes 4, 5, 6, and 8 are negative for the β17 mutation as only 190-bp amplified products are seen. Lanes 7 is homozygote for the β17 mutation as only 170-bp amplified products is seen. Lanes 1, 2, and 3 are heterozygote for the β17 mutation since both 170 and 190-bp amplified products are seen.*

**Detection of amplified products**: The amplified products were separated in 2.5% agarose gel electrophoresis at 120 V for 15–20 min before visualizing with a UV-transilluminator. The fragments sizing 170 and 190 bp indicate presence and

**Interpretation**: Samples having only 170-bp amplified fragments are homozy-

/β<sup>T</sup>

17 with genotype of either β

types of β-globin gene mutation). Samples having both 170 and 190-bp amplified

**Procedure**: The 25-μL PCR is performed containing 250 ng genomic DNA, 200 μM of each dNTP; 0.5 units Taq DNA polymerase, 100 ng of "Common primer"

; 5′-CGT GGA TGA AGT TGG TGG AG-3′, 150 ng of "Mutant primer" for β<sup>E</sup>

5′-CTG CCC TGT GGG CAA GGT GAA CGT GGA TGA AGT TGG TGG AA-3′,

**Thermal cycles**: A total of 35 thermal cycles was carried out with each cycle comprising DNA denaturation at 94°C for 1 min, primer annealing at 56°C for 1 min and primer extension at 72°C for 1 min; the initial denaturation was extended to

**Detection of amplified products**: The amplified products were separated in 2.5% agarose gel electrophoresis at 120 V for 15–20 min before visualizing with a UV-transilluminator. The fragments sizing 160 and 138 bp indicate presence and

**Interpretation**: Samples having only 160-bp amplified fragments are homozy-

mutation, respectively (**Figure 24**).

/β<sup>T</sup>

; 5′-GGC AGA GAG AGT CAG TGC CTA-3′, 100 ng of "Normal primer" for

17. Samples having only 190-bp fragments are nega-

. Samples having only 138-bp fragments are negative

(A represents HbA; T represents other types

(A represents HbA; T represents other

17/βA or β

17/β<sup>T</sup> (A

;

17; 5′-ACC TGA CTC CTG AGG AGA AGA CTG CCG TTA CTG CCC

17; 5′-TCT GCC GTT ACT GCC

**148**

*MS-PCR for identifying β<sup>E</sup> mutation. Lane 1 is negative for the βE mutation as only 138-bp amplified products is seen. Lane 2 is heterozygote for βE mutation as both 138-bp and 160-bp amplified products are seen.*

of β-globin gene mutation). Samples having both 138 and 160-bp amplified products are heterozygote for the β<sup>E</sup> with genotype of either β<sup>E</sup> /βA or β<sup>E</sup> /β<sup>T</sup> (A represents HbA; T represents other types of β-globin gene mutation).
