**2. Screening laboratory tests for β-thalassemia and HbE carriers**

Two laboratory tests are usually performed for screening of the β-thalassemia carriers: automated red cell indices (mean corpuscular volume; MCV, mean corpuscular hemoglobin; MCH, and red cell distribution width; RDW) and one-tube osmotic fragility test (OFT). The screening tests for HbE carrier comprise those performed for the β-thalassemia screen plus the tests used for HbE screen. These tests include dichlorophenolindophenol precipitation (DCIP) test [40], HbE-tube test [15], and hemoglobin E test [16]. The results of these screening tests indicate chance that the blood samples are carriers of either β-thalassemia or HbE.

**127**

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

Red blood cells or erythrocyte indices used for screening for β-thalassemia carriers and HbE carriers conventionally included mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH). These red cell indices must be obtained by automated blood cell analyzers. Normal ranges of MCV and MCH are 85.5 ± 6.8 fL and 27.1 ± 3.1 pg, respectively. Cut-off points of MCV and MCH are 80 fL and 27 pg,

MCV and MCH in β-thalassemia carriers are 68.7 ± 5.4 fL and 20.6 ± 2.1 pg. In HbE carriers, MCV and MCH values are 76.3 ± 4.6 fL and 24.2 ± 1.5 pg [41]. We found that the MCV and MCH in normal individuals, β-thalassemia carriers, and HbE carriers were significantly different [41]. With the cut-off points of 80 fL, MCV has been shown to be effective in screening for the β-thalassemia carriers with sensitivity and specificity of 92.9 and 83.9%, respectively [42, 43]. At 26.5 pg cut-off point of MCH, Pranpanus and co-workers found 92.5% sensitivity and 83.2% specificity of MCH in screening for the β-thalassemia carriers. At the cut-off point of 80 fL for MCV and 27 pg for MCH, Karimi and co-workers found 98.5% sensitivity of MCH, which was more than that of MCV (97.6% sensitivity) in screening for β-thalassemia carriers. They concluded that the MCH was better than

MCV and MCH are not effective in screening for HbE carriers since mean levels in HbE carriers are just slightly lower than those in normal individuals, but the distribution overlaps substantially. Yeo et al. showed that the use of 80 fL cut-off point could miss cases of HbE carriers [45]. Ittarat et al. showed that 5% of HbE carriers would be missed in 80-fL cut-off points of MCV was used [46]. This group suggested the use of some discriminant functions, F1 = 0.01 × MCH × (MCV) or F2 = RDW × MCH × 2(MCV)/Hb × 100 or F3 = MCV/RBC, to increase effectiveness of using red cell indices for screening for HbE carriers. Our unpublished data showed that using 80-fL and 27 pg cut-off points of MCV and MCH, respectively, did not miss cases HbE carriers regardless

This simple test utilizes osmosis, the movement of water from lower to higher salt concentration region, to test for the osmotic resistance of the red blood cells. In a hypotonic condition, the fixed concentration of salt on the outside is lower than that on the inside of red blood cell, resulting in net water movement into the red blood cell. Normal red blood cells are then lysed and the mixture then turns reddish and clear. Red blood cells of the β-thalassemia and HbE carriers have higher osmotic resistance and thus have slower rupture rate, and the mixture remains turbid. Different laboratories may be using slightly different recipes for preparation of hypotonic salt solution such as 0.36% NaCl in distilled water (DW), 0.36% NaCl in phosphate buffer or buffered saline solution (BSS), and 0.45% NaCl in glycerine or glycerine saline solution (GSS). All of these solutions are normally based on the

By mixing the 20 μL of red blood cells of normal and β-thalassemia carriers in 2 mL of 0.36% NaCl in DW (0.36 g NaCl dissolved in 100 mL DW) or BSS (0.32 g NaCl, 0.05 g Na2HPO4, 0.01 g NaH2PO4 dissolved in 100 mL DW) and leave for 5 min, the normal red blood cells will completely lyse and the mixture turns

**2.1 Red blood cell indices**

respectively.

MCH [44].

of hemoglobin levels (4.2–15.1 g/dL).

**2.2 One-tube osmotic fragility test (OFT)**

same concept of kinetic osmotic fragility.

*2.2.1 0.36% NaCl-based and BSS-based one-tube osmotic fragility test*

### **2.1 Red blood cell indices**

*Beta Thalassemia*

testing.

definitely identify types of thalassemia the patients are suffering. Laboratory data thus help define specific types of thalassemia disease of those affected individuals. For the carriers, as they are clinically normal, clinical data are of no use. Only

Conventionally, the laboratory tests for diagnosis of β-thalassemia and HbE include screening tests and confirmatory tests. Initial screening tests are defined as techniques that are simple and relatively low cost, which can indicate the possibility of having thalassemia. These tests should involve the least sample pretreatment and rapid sample preparation and may not need special instrumentation. This would lead to low cost and high sample throughput analysis. The screening tests, however, cannot provide the information on the exact type of thalassemia of the positive persons. Positive samples need further confirmatory test while negative samples can be eliminated from further complicated and expensive

The screening tests for β-thalassemia carriers comprise one-tube osmotic fragility test (OFT) and automated red blood cell indices (mean corpuscular volume; MCV, mean corpuscular hemoglobin; MCH, and red cell distribution width; RDW). Screening tests for HbE carriers are composed of all tests performed to screen for the β-thalassemia carrier plus the specifically established for HbE screen such as dichlorophenolindophenol precipitation (DCIP) test or HbE tube test or HbE test [13–16]. These screening tests, however, cannot provide the information on the

The purpose and methodologies of confirmatory tests for β-thalassemia and HbE are identical. The confirmatory tests must be highly specific in order to obtain the correct diagnosis of carriers of β-thalassemia and HbE as well as the disease state of HbE/β-thalassemia and homozygote or compound heterozygote of the

• Hemoglobin studies: Tests for hemoglobin studies include cellulose acetate electrophoresis, microcolumn chromatography, alkaline denaturation test, cation-exchange high performance liquid chromatography (HPLC) [17–19], cation-exchange low pressure liquid chromatography (LPLC) [20–22], capillary zone electrophoresis (CZE) [23–27], sandwich enzyme linked immunosorbent assay (ELISA) for Hb F [28], Hb Bart's [29] and Hb A2 [30], flow cytometric analysis of F cells [31], and immunochromatographic strip (IC

• DNA analysis: Tests for analysis of β-globin gene mutations include multiplex allele-specific polymerase chain reaction (MAS-PCR) [29], amplification refractory mutation system (ARMS)-PCR [33], mutagenically separated (MS)- PCR [34–36], and high resolution melting curve (HRM) analysis [37–39].

Two laboratory tests are usually performed for screening of the β-thalassemia carriers: automated red cell indices (mean corpuscular volume; MCV, mean corpuscular hemoglobin; MCH, and red cell distribution width; RDW) and one-tube osmotic fragility test (OFT). The screening tests for HbE carrier comprise those performed for the β-thalassemia screen plus the tests used for HbE screen. These tests include dichlorophenolindophenol precipitation (DCIP) test [40], HbE-tube test [15], and hemoglobin E test [16]. The results of these screening tests indicate

**2. Screening laboratory tests for β-thalassemia and HbE carriers**

chance that the blood samples are carriers of either β-thalassemia or HbE.

laboratory data can define β-thalassemia and HbE carriers.

exact type of thalassemia of the positive persons.

β-thalassemia gene. The confirmatory tests include;

strip) test for Hb Bart's [32].

**126**

Red blood cells or erythrocyte indices used for screening for β-thalassemia carriers and HbE carriers conventionally included mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH). These red cell indices must be obtained by automated blood cell analyzers. Normal ranges of MCV and MCH are 85.5 ± 6.8 fL and 27.1 ± 3.1 pg, respectively. Cut-off points of MCV and MCH are 80 fL and 27 pg, respectively.

MCV and MCH in β-thalassemia carriers are 68.7 ± 5.4 fL and 20.6 ± 2.1 pg. In HbE carriers, MCV and MCH values are 76.3 ± 4.6 fL and 24.2 ± 1.5 pg [41]. We found that the MCV and MCH in normal individuals, β-thalassemia carriers, and HbE carriers were significantly different [41]. With the cut-off points of 80 fL, MCV has been shown to be effective in screening for the β-thalassemia carriers with sensitivity and specificity of 92.9 and 83.9%, respectively [42, 43]. At 26.5 pg cut-off point of MCH, Pranpanus and co-workers found 92.5% sensitivity and 83.2% specificity of MCH in screening for the β-thalassemia carriers. At the cut-off point of 80 fL for MCV and 27 pg for MCH, Karimi and co-workers found 98.5% sensitivity of MCH, which was more than that of MCV (97.6% sensitivity) in screening for β-thalassemia carriers. They concluded that the MCH was better than MCH [44].

MCV and MCH are not effective in screening for HbE carriers since mean levels in HbE carriers are just slightly lower than those in normal individuals, but the distribution overlaps substantially. Yeo et al. showed that the use of 80 fL cut-off point could miss cases of HbE carriers [45]. Ittarat et al. showed that 5% of HbE carriers would be missed in 80-fL cut-off points of MCV was used [46]. This group suggested the use of some discriminant functions, F1 = 0.01 × MCH × (MCV) or F2 = RDW × MCH × 2(MCV)/Hb × 100 or F3 = MCV/RBC, to increase effectiveness of using red cell indices for screening for HbE carriers. Our unpublished data showed that using 80-fL and 27 pg cut-off points of MCV and MCH, respectively, did not miss cases HbE carriers regardless of hemoglobin levels (4.2–15.1 g/dL).

### **2.2 One-tube osmotic fragility test (OFT)**

This simple test utilizes osmosis, the movement of water from lower to higher salt concentration region, to test for the osmotic resistance of the red blood cells. In a hypotonic condition, the fixed concentration of salt on the outside is lower than that on the inside of red blood cell, resulting in net water movement into the red blood cell. Normal red blood cells are then lysed and the mixture then turns reddish and clear. Red blood cells of the β-thalassemia and HbE carriers have higher osmotic resistance and thus have slower rupture rate, and the mixture remains turbid. Different laboratories may be using slightly different recipes for preparation of hypotonic salt solution such as 0.36% NaCl in distilled water (DW), 0.36% NaCl in phosphate buffer or buffered saline solution (BSS), and 0.45% NaCl in glycerine or glycerine saline solution (GSS). All of these solutions are normally based on the same concept of kinetic osmotic fragility.

### *2.2.1 0.36% NaCl-based and BSS-based one-tube osmotic fragility test*

By mixing the 20 μL of red blood cells of normal and β-thalassemia carriers in 2 mL of 0.36% NaCl in DW (0.36 g NaCl dissolved in 100 mL DW) or BSS (0.32 g NaCl, 0.05 g Na2HPO4, 0.01 g NaH2PO4 dissolved in 100 mL DW) and leave for 5 min, the normal red blood cells will completely lyse and the mixture turns

#### **Figure 1.**

*0.36% NaCl-based and BSS-based one-tube osmotic fragility test for screening of β-thalassemia and HbE carriers. The β-thalassemia carriers all have the positive OFT results, while HbE carriers have either positive, negative, and suspicious OFT results (see the text for detail).*

reddish-clear and reported as OFT-Negative. In contrast, the mixture of blood samples of β-thalassemia carriers and 0.36% NaCl remains turbid at 5 min and reported as OFT-Positive. In case that the appearance of the mixture is between positive and negative OFT-results, it is reported as OFT-suspicious (**Figure 1**). Chow et al. showed that this test has 95% sensitivity and 86% specificity for screening the β-thalassemia carrier [47]. Bobhate et al. demonstrated 97.1% sensitivity and 100% specificity of this test, which they called NESTROFT, in screening for β-thalassemia carriers [48], while Mamtani et al. showed 93.4% and 97.2% sensitivity and specificity of this test for screening of the β-thalassemia carriers [49]. For HbE carriers, Fucharoen et al. showed 37.7% false negative OFT result in HbE carriers [50], which was closed to our unpublished data that showed approximately 29.4% false negative results.

### *2.2.2 0.45% NaCl in glycerine or glycerine saline solution (GSS)-based one-tube osmotic fragility test*

This test was established by Prof. Dr. Torpong Sa-nguansermsri of Thalassemia Unit, Department of Pediatrics, Faculty of Medicine, Chiang Mai University and named this test "Erythrocyte Osmotic Fragility Test" (EOFT) [51]. In this platform, the influx of water into the erythrocyte is slow and hemolysis can be measured at any points of time after mixing blood with 0.45% GSS [1.424 g Na2HPO4.2H2O, 0.262 g NaH2PO4.2H2O, 2.812 g NaCl, 19.27 g glycerine (87%), and DW to make 1000 mL]. Technically, 10 μL of EDTA blood is mixed with 10 mL of 0.45% GSS and read 620-nm absorbance at 15, 30, 45, 60, and 120 s, before calculating hemolysis rate at every time point.

The cut-off point of hemolysis rate is 60%. Positive blood samples have hemolysis rate <60%, while the blood samples having hemolysis rate ≥60% is judged as negative. The β-thalassemia carriers have hemolysis rate of 17.6 ± 8.1%. Hemolysis rate of 32.6 ± 13.2% is observed in HbE carriers [52, 53].

#### *2.2.3 0.45% NaCl in glycerine or glycerine saline solution (GSS)-based one-tube osmotic fragility test expressed in "hemolysis area"*

The portable spectrophotometer was invented and capable of reading absorbance and transmission of red light through the red blood cell suspension inside

**129**

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

cuvette [54]. To perform this test, 20 μL of EDTA blood is mixed with 5 mL of GSS in 12 × 75 polypropylene cuvette. Then place the cuvette inside the cuvette holder of the portable spectrophotometer (red dots in **Figure 2(A)**) and start the machine. Light transmission (red line in **Figure 2(B)**), light absorbance (black line in **Figure 2(B)**), and HemeArea or hemolysis area (green line in **Figure 2(B)**) are generated simultaneously in real-time manner. At 120 s, the numeric HemeArea

*Portable spectrophotometer (A) and on-screen GSS-based OFT result with the hemolysis area (HemeArea in* 

Cut-off point of the hemolysis area is 52.4 unit. Blood samples having the hemolysis area less than these cut-off values are judged as "positive samples," while those samples having the hemolysis area ≥ 52.4 units are classified as "negative sample." The hemolysis area of normal individuals was 67.1 ± 12.6 units. In contrast, the hemolysis area of HbE carriers, β-thalassemia carriers were found to be 36.4 ± 13.9,

In dichlorophenolindophenol precipitation (DCIP) test, 2,6-dichiorophenol indophenol (DCIP) were oxidizing chemicals and used as indicator of ascorbic acid measurement. Hemoglobin E is resulted from amino acid change at codon 26 of β-globin chain from glutamic acid to lysine. This change makes contact of

HbE changes from tetramer to monomer, freeing sulfhydryl group of amino acid,

The 500-mL DCIP reagent is composed of 4.36 g Trizma base, 2.68 g EDTA-Na2.2H2O, 0.0276 g of DCIP, and 0.05 g of saponin. The pH of reagent is adjusted to 7.5 by using 6 N HCl. To perform test, 20 μL of EDTA blood is mixed with 5 mL DCIP reagent. The mixture is incubated in 37°C-water bath for 1 h before precipitation occurs in case of HbE carriers. To enhance visualization, 20 μL of 6% (w/v) ascorbic acid is dropped into the mixture and the color of mixture turns from deep

This test is now commercially available in Thailand. The commercial DCIP reagent has the same ingredient and incubation condition as the original test, except the volume of reagent is scaled down to 2 mL. The examples of commercially DCIP reagent presently distributed in Thailand are THALCON™ and KKU-DCIP™


(or hemolysis area) is shown in green alphabets (**Figure 2(B)**).

**2.3 Dichlorophenolindophenol precipitation (DCIP) test**

oxidized by the DCIP, denatured, and precipitated [55].

18.6 ± 1.1, respectively [54].

*the screen) shown in the screen (B).*

**Figure 2.**

α-globin chain and β<sup>E</sup>

blue to pale red (**Figure 3**).

reagent (**Figure 4**).

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

**Figure 2.**

*Beta Thalassemia*

**Figure 1.**

29.4% false negative results.

*negative, and suspicious OFT results (see the text for detail).*

*osmotic fragility test*

sis rate at every time point.

reddish-clear and reported as OFT-Negative. In contrast, the mixture of blood samples of β-thalassemia carriers and 0.36% NaCl remains turbid at 5 min and reported as OFT-Positive. In case that the appearance of the mixture is between positive and negative OFT-results, it is reported as OFT-suspicious (**Figure 1**). Chow et al. showed that this test has 95% sensitivity and 86% specificity for screening the β-thalassemia carrier [47]. Bobhate et al. demonstrated 97.1% sensitivity and 100% specificity of this test, which they called NESTROFT, in screening for β-thalassemia carriers [48], while Mamtani et al. showed 93.4% and 97.2% sensitivity and specificity of this test for screening of the β-thalassemia carriers [49]. For HbE carriers, Fucharoen et al. showed 37.7% false negative OFT result in HbE carriers [50], which was closed to our unpublished data that showed approximately

*0.36% NaCl-based and BSS-based one-tube osmotic fragility test for screening of β-thalassemia and HbE carriers. The β-thalassemia carriers all have the positive OFT results, while HbE carriers have either positive,* 

*2.2.2 0.45% NaCl in glycerine or glycerine saline solution (GSS)-based one-tube* 

This test was established by Prof. Dr. Torpong Sa-nguansermsri of Thalassemia Unit, Department of Pediatrics, Faculty of Medicine, Chiang Mai University and named this test "Erythrocyte Osmotic Fragility Test" (EOFT) [51]. In this platform, the influx of water into the erythrocyte is slow and hemolysis can be measured at any points of time after mixing blood with 0.45% GSS [1.424 g Na2HPO4.2H2O, 0.262 g NaH2PO4.2H2O, 2.812 g NaCl, 19.27 g glycerine (87%), and DW to make 1000 mL]. Technically, 10 μL of EDTA blood is mixed with 10 mL of 0.45% GSS and read 620-nm absorbance at 15, 30, 45, 60, and 120 s, before calculating hemoly-

The cut-off point of hemolysis rate is 60%. Positive blood samples have hemolysis rate <60%, while the blood samples having hemolysis rate ≥60% is judged as negative. The β-thalassemia carriers have hemolysis rate of 17.6 ± 8.1%. Hemolysis

*2.2.3 0.45% NaCl in glycerine or glycerine saline solution (GSS)-based one-tube* 

The portable spectrophotometer was invented and capable of reading absorbance and transmission of red light through the red blood cell suspension inside

rate of 32.6 ± 13.2% is observed in HbE carriers [52, 53].

*osmotic fragility test expressed in "hemolysis area"*

**128**

*Portable spectrophotometer (A) and on-screen GSS-based OFT result with the hemolysis area (HemeArea in the screen) shown in the screen (B).*

cuvette [54]. To perform this test, 20 μL of EDTA blood is mixed with 5 mL of GSS in 12 × 75 polypropylene cuvette. Then place the cuvette inside the cuvette holder of the portable spectrophotometer (red dots in **Figure 2(A)**) and start the machine. Light transmission (red line in **Figure 2(B)**), light absorbance (black line in **Figure 2(B)**), and HemeArea or hemolysis area (green line in **Figure 2(B)**) are generated simultaneously in real-time manner. At 120 s, the numeric HemeArea (or hemolysis area) is shown in green alphabets (**Figure 2(B)**).

Cut-off point of the hemolysis area is 52.4 unit. Blood samples having the hemolysis area less than these cut-off values are judged as "positive samples," while those samples having the hemolysis area ≥ 52.4 units are classified as "negative sample." The hemolysis area of normal individuals was 67.1 ± 12.6 units. In contrast, the hemolysis area of HbE carriers, β-thalassemia carriers were found to be 36.4 ± 13.9, 18.6 ± 1.1, respectively [54].

#### **2.3 Dichlorophenolindophenol precipitation (DCIP) test**

In dichlorophenolindophenol precipitation (DCIP) test, 2,6-dichiorophenol indophenol (DCIP) were oxidizing chemicals and used as indicator of ascorbic acid measurement. Hemoglobin E is resulted from amino acid change at codon 26 of β-globin chain from glutamic acid to lysine. This change makes contact of α-globin chain and β<sup>E</sup> -globin chain less stable. Thus, in DCIP solution, molecule of HbE changes from tetramer to monomer, freeing sulfhydryl group of amino acid, oxidized by the DCIP, denatured, and precipitated [55].

The 500-mL DCIP reagent is composed of 4.36 g Trizma base, 2.68 g EDTA-Na2.2H2O, 0.0276 g of DCIP, and 0.05 g of saponin. The pH of reagent is adjusted to 7.5 by using 6 N HCl. To perform test, 20 μL of EDTA blood is mixed with 5 mL DCIP reagent. The mixture is incubated in 37°C-water bath for 1 h before precipitation occurs in case of HbE carriers. To enhance visualization, 20 μL of 6% (w/v) ascorbic acid is dropped into the mixture and the color of mixture turns from deep blue to pale red (**Figure 3**).

This test is now commercially available in Thailand. The commercial DCIP reagent has the same ingredient and incubation condition as the original test, except the volume of reagent is scaled down to 2 mL. The examples of commercially DCIP reagent presently distributed in Thailand are THALCON™ and KKU-DCIP™ reagent (**Figure 4**).

#### **Figure 3.**

*Dichlorophenolindophenol precipitation (DCIP) test. The DCIP results before adding ascorbic acid have deeply blue color, but after ascorbic acid addition, the color turns to pale red. The amount of precipitation is increased in homozygous HbE (EE), compared to HbE carrier (AE). No precipitation is seen for non-HbE (A2A). Modified from Ref. [53].*

#### **Figure 4.**

*DCIP test results obtained by commercial DCIP kit. The black lines placed behind the tube aid the result reading. If we cannot see the black lines, the result is positive. If the black lines are clearly seen, the result is negative. DCIP test produces turbid solution.*

This test has been validated and shown to be effective in screening for HbE carriers. Analysis in the author's laboratory showed that this test had 100% sensitivity and 98.4% specificity in screening for HbE carriers [56]. Wiwanitkit et al. found almost the same effectiveness of this test in HbE screen; 100% sensitivity and 97.2% specificity [14]. Chapple et al. re-evaluated the KKU-DCIP reagent kit and found 100% sensitivity and 92% specificity for screening for HbE carriers [55].

#### **2.4 HbE-tube test**

This test was invented by the author in 2012 [15]. It is based on the principle of anion-exchange liquid chromatography. The diethyl aminoethyl (DEAE)-cellulose having positive charge in buffer having fixed amount of NaCl is placed in the test tube. This condition allows all hemoglobins to bind DEAE-cellulose, except HbE and HbA2. Then these two hemoglobins will still dissolve in the supernatant. However, since HbE quantity is more than 10% in HbE carriers, then in case of HbE carrier, the supernatant color is red. In contrast, in non HbE, that is, no HbE in blood, the supernatant is colorless (**Figure 5**). This test is simple, requiring no incubation and centrifugation. Standing the test tube after mixing the blood for about 10 min is enough for visualizing the supernatant color.

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

the color of supernatant as shown in **Figure 5**.

(2–8°C) for 5 months [15].

*A2A and colorless supernatant is seen.*

**2.5 HbE screen test**

**Figure 5.**

cation [59, 60].

be colorless (**Figure 6**).

To perform the test, 15 μL packed red cell (PRC) is added to pre-prepared DEAE-cellulose suspension in the transparent test tube (0.5 mL DEAE-cellulose suspension in 1.0 mL glycine-NaCl buffer containing 0.2 M glycine–0.01% KCN– 7.5 mM NaCl). The tube is shaken briefly, left for 5 min at room temperature, and spun at 3500 round per minute for 5 min before reading the results by visualizing

*HbE tube test for screening of HbE carriers. The positive tube (Pos) is the result of HbE carrier having Hb type AE and red supernatant is seen. The negative tube (Neg) is the result of normal individual having Hb type* 

This test was simple and effective in screening of HbE carriers. Its sensitivity and specificity for HbE screen are 100%. The reagent can be kept in the cold

This test was initially invented by Prof. Dr. Torpong Sa-nguansermsri of The Thalassemia Unit, Department of Pediatrics, Faculty of Medicine, Chiang Mai University, Thailand [57] and adopted for HbE screen in pregnant women by Sirichotiyakul et al. [58]. This test was also established in author's laboratory (Unpublished data). This test works under the principle of anion-exchange column chromatography, modified from microcolumn chromatography for HbA2 quantifi-

The test comprises small syringe packed inside with anion-coated resin such as diethylaminoethyl (DEAE)-Sephadex A50 suspended in Tris-HCl-KCN (THK) buffer (6.057 g tris hydroxymethyl aminomethane, 0.1 g KCN, 4 N HCl to adjust pH), pH 8.5. On passing hemolysate through this microcolumn, all hemoglobins bind to the resin. However, passing the eluting buffer (THK buffer, pH 8.2), only HbE and HbA2 are eluted. If the patients have HbE, the color of entire length of the microcolumn will be red. If the patients do not have HbE, the color of this point will

To perform this test invented by Prof. Dr. Torpong Sanguansermsri, 10 mL hemoglobin solution (40 μL hemolysate mixed in 5 mL THK buffer pH 8.5) is dropped into microcolumn prepacked with DEAE-Sephadex A 50 to the height of 5.0 cm in pasture pipette. The solution is allowed to flow through the microcolumn which is subsequently equilibrated with 10 mL THK buffer pH 8.5. Finally, HbE and HbA2 are eluted out of the microcolumn after poring 10 mL of THK buffer, pH 8.2. At the end, the red color of the column is observed. If almost the entire length of microcolumn is red,

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

#### **Figure 5.**

*Beta Thalassemia*

**130**

**2.4 HbE-tube test**

*negative. DCIP test produces turbid solution.*

**Figure 4.**

**Figure 3.**

*(A2A). Modified from Ref. [53].*

This test has been validated and shown to be effective in screening for HbE carriers. Analysis in the author's laboratory showed that this test had 100% sensitivity and 98.4% specificity in screening for HbE carriers [56]. Wiwanitkit et al. found almost the same effectiveness of this test in HbE screen; 100% sensitivity and 97.2% specificity [14]. Chapple et al. re-evaluated the KKU-DCIP reagent kit and found

*DCIP test results obtained by commercial DCIP kit. The black lines placed behind the tube aid the result reading. If we cannot see the black lines, the result is positive. If the black lines are clearly seen, the result is* 

*Dichlorophenolindophenol precipitation (DCIP) test. The DCIP results before adding ascorbic acid have deeply blue color, but after ascorbic acid addition, the color turns to pale red. The amount of precipitation is increased in homozygous HbE (EE), compared to HbE carrier (AE). No precipitation is seen for non-HbE* 

This test was invented by the author in 2012 [15]. It is based on the principle of anion-exchange liquid chromatography. The diethyl aminoethyl (DEAE)-cellulose having positive charge in buffer having fixed amount of NaCl is placed in the test tube. This condition allows all hemoglobins to bind DEAE-cellulose, except HbE and HbA2. Then these two hemoglobins will still dissolve in the supernatant. However, since HbE quantity is more than 10% in HbE carriers, then in case of HbE carrier, the supernatant color is red. In contrast, in non HbE, that is, no HbE in blood, the supernatant is colorless (**Figure 5**). This test is simple, requiring no incubation and centrifugation. Standing the test tube after mixing the blood for

100% sensitivity and 92% specificity for screening for HbE carriers [55].

about 10 min is enough for visualizing the supernatant color.

*HbE tube test for screening of HbE carriers. The positive tube (Pos) is the result of HbE carrier having Hb type AE and red supernatant is seen. The negative tube (Neg) is the result of normal individual having Hb type A2A and colorless supernatant is seen.*

To perform the test, 15 μL packed red cell (PRC) is added to pre-prepared DEAE-cellulose suspension in the transparent test tube (0.5 mL DEAE-cellulose suspension in 1.0 mL glycine-NaCl buffer containing 0.2 M glycine–0.01% KCN– 7.5 mM NaCl). The tube is shaken briefly, left for 5 min at room temperature, and spun at 3500 round per minute for 5 min before reading the results by visualizing the color of supernatant as shown in **Figure 5**.

This test was simple and effective in screening of HbE carriers. Its sensitivity and specificity for HbE screen are 100%. The reagent can be kept in the cold (2–8°C) for 5 months [15].

#### **2.5 HbE screen test**

This test was initially invented by Prof. Dr. Torpong Sa-nguansermsri of The Thalassemia Unit, Department of Pediatrics, Faculty of Medicine, Chiang Mai University, Thailand [57] and adopted for HbE screen in pregnant women by Sirichotiyakul et al. [58]. This test was also established in author's laboratory (Unpublished data). This test works under the principle of anion-exchange column chromatography, modified from microcolumn chromatography for HbA2 quantification [59, 60].

The test comprises small syringe packed inside with anion-coated resin such as diethylaminoethyl (DEAE)-Sephadex A50 suspended in Tris-HCl-KCN (THK) buffer (6.057 g tris hydroxymethyl aminomethane, 0.1 g KCN, 4 N HCl to adjust pH), pH 8.5. On passing hemolysate through this microcolumn, all hemoglobins bind to the resin. However, passing the eluting buffer (THK buffer, pH 8.2), only HbE and HbA2 are eluted. If the patients have HbE, the color of entire length of the microcolumn will be red. If the patients do not have HbE, the color of this point will be colorless (**Figure 6**).

To perform this test invented by Prof. Dr. Torpong Sanguansermsri, 10 mL hemoglobin solution (40 μL hemolysate mixed in 5 mL THK buffer pH 8.5) is dropped into microcolumn prepacked with DEAE-Sephadex A 50 to the height of 5.0 cm in pasture pipette. The solution is allowed to flow through the microcolumn which is subsequently equilibrated with 10 mL THK buffer pH 8.5. Finally, HbE and HbA2 are eluted out of the microcolumn after poring 10 mL of THK buffer, pH 8.2. At the end, the red color of the column is observed. If almost the entire length of microcolumn is red,

#### **Figure 6.**

*HbE screening test in a modified protocol so-called naked eye microcolumn for HbE screen (NEMES). The AE sample shows red color in the resin along entire length of the resin, but not for the A2A sample.*

the result is "Positive." However, if the red color sticks to only at the top layer of the packed resin, the result is "Negative" [57]. This test was evaluated by Sirichotiyakul et al. and found to have 100% sensitivity and 100% specificity for HbE screen [58].

Alternatively, the test was modified in author's laboratory. In this modification, DEAE-cellulose was used as the pre-packed resin, and glycine-NaCl buffer was equilibrating and eluting buffer. To prepare microcolumn, DEAE-cellulose resin suspended in equilibrating buffer (0.2 M glycine + 0.01% KCN) is pre-packed to the height of 2.5 cm in 3-mL plastic syringe with 1-cm diameter. To perform the test, 150 μL hemolysate is dropped into the microcolumn and allowed to pass through the resin before eluting HbE with 4 mL eluting buffer (0.2 M glycine + 0.01% KCN + 0.005 M NaCl). The red color of the resin packed in column is observed in the way resemble that mentioned above (**Figure 6**). The protocol was named "Naked-EyE-Microcolumn-HbE-Screen or NEMES" [61]. This test was found to have 100% sensitivity and 100% specificity for screening of HbE carriers.

#### **3. Interpretation of the screening tests**

The β-thalassemia carriers always have positive OFT, MCV less than 80 fL, MCH less than 27 pg, and negative HbE. In contrast, HbE carriers may have MCV: more or less than 80 fL, MCH: more or less than 27 pg, OFT: positive or suspicious or negative, and hemolysis area: more or less than 52.4 unit. However, all cases of HbE carriers certainly have positive HbE screening tests performed by all mentioned tests.

The MCV, MCH, and OFT are all positive in β-thalassemia diseases such as homozygous βO-thalassemia, homozygous β<sup>+</sup> -thalassemia, compound heterozygous βO/β<sup>+</sup> -thalassemia, compound heterozygous HbE/βO-thalassemia, compound heterozygous HbE/β<sup>+</sup> -thalassemia, and homozygous HbE. However, the DCIP test, HbE tube test, and HbE screen test are positive in all cases having HbE in blood.

#### **4. Usefulness of screening tests**

In financially burden countries that have considerably high prevalence of thalassemia and hemoglobinopathies, initial screen of the carriers in population is essential. This approach helps to decrease the number of cases seeking further for more

**133**

**Figure 7.**

*pattern on CAE at pH 8.6 stained with Ponceau S stain.*

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

hemoglobin studies and DNA analysis.

*condition*

expensive confirmatory tests. Normally, the sophisticated laboratory tests for conforming the diagnosis of thalassemia and hemoglobinopathies are set up in big centers which are mostly located in the city. Thus, selected cases that are screened in for the definite diagnosis of thalassemia and hemoglobinopathies have to travel a long distance to the city. This would not consume much money for traveling and for laboratory tests.

Aims of confirmatory tests are to make the definite diagnosis of β-thalassemia and HbE. These tests generally performed only in blood samples having positive results of screening tests. Two sets of confirmatory tests are generally performed;

**4.1 Confirmatory method for thalassemia and hemoglobinopathies**

*4.1.1 Hemoglobin studies by cellulose acetate electrophoresis (CAE) at alkaline* 

(Tris), 1.22 g ethylenediaminetetraacetic acid (EDTA), and 1.5 g boric acid. The electrophoresis is performed at a constant voltage of 250–300 volts for 15–20 minutes or until HbA and HbE bands are clearly separated. At the end of electrophoresis, the hemoglobin bands on cellulose acetate plate are stained for 5 minutes with Ponceau S stain (2 g of Ponceau S powder, 30 g of trichloroacetic acid, and 30 g of sulfosalicylic acid in 1 L of distilled water) and destained for 5 minutes in destaining solution (5% acetic acid in distilled water). Finally, the cellulose acetate plate is made clear for permanent record by immersing for 5 minutes in clearing solution (4 volume of methanol +1 volume of glacial acetic acid) and dried by using hair dryer. Hemoglobin pattern from cathodic to anodic ends is Constant Spring-A2/E/C/O-S/D/Lepore/G-F-A-Portland-Bart's-H [64] (**Figure 7**). Hemoglobin patterns on CAE at alkaline condition are shown in **Table 1**. In the past, densitometer was used to determine quantities of hemoglobins in blood samples. However, this technique is not conventionally performed presently because it may give falsely high levels of hemoglobins if the background is not completely cleared. However, CAE results can still give types of hemoglobins in blood samples. Thus, by

*(A) Hemoglobin patterns on cellulose acetate electrophoresis (CAE) at pH 8.6. (B) Example of hemoglobin* 

This test separates hemoglobins in blood by their negatively molecular net charge. Hemoglobins are allowed to dissolve in Tris-EDTA-Borate (TBE) buffer pH 8.6. This pH is more than isoelectric points (pI) of all hemoglobins (approximately 6.5–7.5) [62, 63]. At this pH, all hemoglobins have negative charge and migrate from cathodic part toward anodic end of the electrophoretic chamber.

In 1 L of TBE buffer, it is composed of 12.0 g tris-hydroxymethyl aminomethane

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

*Beta Thalassemia*

**Figure 6.**

the result is "Positive." However, if the red color sticks to only at the top layer of the packed resin, the result is "Negative" [57]. This test was evaluated by Sirichotiyakul et al. and found to have 100% sensitivity and 100% specificity for HbE screen [58]. Alternatively, the test was modified in author's laboratory. In this modification, DEAE-cellulose was used as the pre-packed resin, and glycine-NaCl buffer was equilibrating and eluting buffer. To prepare microcolumn, DEAE-cellulose resin suspended in equilibrating buffer (0.2 M glycine + 0.01% KCN) is pre-packed to the height of 2.5 cm in 3-mL plastic syringe with 1-cm diameter. To perform the test, 150 μL hemolysate is dropped into the microcolumn and allowed to pass through the resin before eluting HbE with 4 mL eluting buffer (0.2 M glycine + 0.01% KCN + 0.005 M NaCl). The red color of the resin packed in column is observed in the way resemble that mentioned above (**Figure 6**). The protocol was named "Naked-EyE-Microcolumn-HbE-Screen or NEMES" [61]. This test was found to have 100% sensitivity and 100% specificity for screening of HbE carriers.

*sample shows red color in the resin along entire length of the resin, but not for the A2A sample.*

*HbE screening test in a modified protocol so-called naked eye microcolumn for HbE screen (NEMES). The AE* 

The β-thalassemia carriers always have positive OFT, MCV less than 80 fL, MCH less than 27 pg, and negative HbE. In contrast, HbE carriers may have MCV: more or less than 80 fL, MCH: more or less than 27 pg, OFT: positive or suspicious or negative, and hemolysis area: more or less than 52.4 unit. However, all cases of HbE carriers certainly have positive HbE screening tests performed by all mentioned tests. The MCV, MCH, and OFT are all positive in β-thalassemia diseases such as


In financially burden countries that have considerably high prevalence of thalassemia and hemoglobinopathies, initial screen of the carriers in population is essential. This approach helps to decrease the number of cases seeking further for more

HbE tube test, and HbE screen test are positive in all cases having HbE in blood.



**3. Interpretation of the screening tests**

homozygous βO-thalassemia, homozygous β<sup>+</sup>

**4. Usefulness of screening tests**

**132**

βO/β<sup>+</sup>

heterozygous HbE/β<sup>+</sup>

expensive confirmatory tests. Normally, the sophisticated laboratory tests for conforming the diagnosis of thalassemia and hemoglobinopathies are set up in big centers which are mostly located in the city. Thus, selected cases that are screened in for the definite diagnosis of thalassemia and hemoglobinopathies have to travel a long distance to the city. This would not consume much money for traveling and for laboratory tests.

#### **4.1 Confirmatory method for thalassemia and hemoglobinopathies**

Aims of confirmatory tests are to make the definite diagnosis of β-thalassemia and HbE. These tests generally performed only in blood samples having positive results of screening tests. Two sets of confirmatory tests are generally performed; hemoglobin studies and DNA analysis.

#### *4.1.1 Hemoglobin studies by cellulose acetate electrophoresis (CAE) at alkaline condition*

This test separates hemoglobins in blood by their negatively molecular net charge. Hemoglobins are allowed to dissolve in Tris-EDTA-Borate (TBE) buffer pH 8.6. This pH is more than isoelectric points (pI) of all hemoglobins (approximately 6.5–7.5) [62, 63]. At this pH, all hemoglobins have negative charge and migrate from cathodic part toward anodic end of the electrophoretic chamber.

In 1 L of TBE buffer, it is composed of 12.0 g tris-hydroxymethyl aminomethane (Tris), 1.22 g ethylenediaminetetraacetic acid (EDTA), and 1.5 g boric acid.

The electrophoresis is performed at a constant voltage of 250–300 volts for 15–20 minutes or until HbA and HbE bands are clearly separated. At the end of electrophoresis, the hemoglobin bands on cellulose acetate plate are stained for 5 minutes with Ponceau S stain (2 g of Ponceau S powder, 30 g of trichloroacetic acid, and 30 g of sulfosalicylic acid in 1 L of distilled water) and destained for 5 minutes in destaining solution (5% acetic acid in distilled water). Finally, the cellulose acetate plate is made clear for permanent record by immersing for 5 minutes in clearing solution (4 volume of methanol +1 volume of glacial acetic acid) and dried by using hair dryer. Hemoglobin pattern from cathodic to anodic ends is Constant Spring-A2/E/C/O-S/D/Lepore/G-F-A-Portland-Bart's-H [64] (**Figure 7**). Hemoglobin patterns on CAE at alkaline condition are shown in **Table 1**. In the past, densitometer was used to determine quantities of hemoglobins in blood samples. However, this technique is not conventionally performed presently because it may give falsely high levels of hemoglobins if the background is not completely cleared. However, CAE results can still give types of hemoglobins in blood samples. Thus, by

#### **Figure 7.**

*(A) Hemoglobin patterns on cellulose acetate electrophoresis (CAE) at pH 8.6. (B) Example of hemoglobin pattern on CAE at pH 8.6 stained with Ponceau S stain.*


**Table 1.**

*Hemoglobin pattern on CAE at pH 8.6 of β-thalassemia and HbE.*

using this technique, further tests must be done in order to determine the level of HbA2 and HbF. Raised level of HbA2 beyond normal range is the diagnostic marker for β-thalassemia carriers, while elevated level of HbF helps identify the high HbF condition found in the hereditary persistence of fetal hemoglobin (HPFH).

#### *4.1.2 Hemoglobin study by microcolumn chromatography*

This test is preliminarily aimed to quantify HbA2 levels that help diagnosis of the β-thalassemia carrier. However, HbE has the same pI as HbA2, thus these two hemoglobins are co-eluted. Microcolumn chromatography is an anion-exchange chromatography-based method. Anion-resin such as DEAE-cellulose or DEAE-Sephadex A50 suspended in appropriate buffer is packed in the microcolumn. On passing hemolysate through the packed resin, negatively charged hemoglobins binds to the resin at different binding affinity. HbA2 and HbE bind to the resin at the weakest strength (if there is no Hb Constant Spring) and are eluted out easily with small amount of external anion such as CL<sup>−</sup>.

There are two types of microcolumn chromatography, based on the anion-resin and buffers used. These include (1) DEAE-Sephadex A50 plus Tris-HCl-KCN buffer, and (2) DEAE-cellulose plus glycine-NaCl buffer.

#### *4.1.2.1 Microcolumn chromatography using DEAE-Sephadex A50 resin*

DEAE-Sephadex A 50 resin suspended in Tris-HCl-KCN buffer (0.05 M Tris plus 0.1 g KCN/1 L, adjust pH with 4 N HCl) pH 8.5 is packed in pasture pipette to the height of 8–9 cm [52]. Then, the microcolumn is applied with 100 μL hemolysate, equilibrated with 10 mL THK buffer pH 8.5. Finally, 10 mL THK buffer pH 8.2 is applied to the microcolumn to elute HbA2 and/or HbE before 10 mL eluate is collected for measuring light absorbance (A) or optical density (OD) at 415 nm. This is then called A415-A2 or OD415-A2. For measuring OD415 of total hemoglobin or OD415-Total Hb, 100 μL hemolysate is mixed with 10 mL DW before measuring the absorbance. The level of HbA2 or HbE is calculated by the Eq. 1 shown below.

$$\text{HbA}\_2 \text{ or } \text{HbE (\%)} = \left[ \text{OD}\_{415} - \text{A}\_2/\text{OD}\_{415} - \text{total Hb} \right] \times 100 \tag{1}$$

**135**

sulphate.

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

If percent is less than 10, it is HbA2. If percent is 10 up, it is HbE.

measuring optical density at 540 nm.

tory in 2007 [65].

*4.1.2.2 Microcolumn chromatography using DEAE-cellulose*

This protocol was described by Wood [64] and modified in the author's labora-

In modified protocol, DEAE-cellulose resin suspended in equilibrating buffer (0.2 M glycine + 0.01% KCN) is packed to the height of 2 cm in plastic microcolumn with 1.0-cm diameter. Then the microcolumn is applied by 50 μL hemolysate (prepared by mixing 1 part of PRC and 6 parts of 0.05% Triton X-100 as hemolysis buffer) and flushed with 5 mL eluting buffer (0.2 M glycine +0.01% KCN + 0.005 M NaCl). The 5-mL eluate is then collected and measured for absorbance or optical density at 415 nm (A415-A2 or OD415-A2). OD415 of total hemoglobin is measured in diluted hemolysate (50 μL hemolysate mixed with DW to 15 mL) and called OD415-Total. Calculation of levels of HbA2 or HbE must follow the Eq. 2 shown below.

HbA2 or HbE (%) = [OD415 − A2/ OD415–total × 3] × 100 (2)

For both protocols, if HbA2 level is less than 3.5%, the chance of β-thalassemia carrier is excluded. Instead, the cases may be either normal of α-thalassemia carriers. However, if HbA2 level is between 3.5 and 10.0%, the case is definitely β-thalassemia carrier. In β-thalassemia carriers, mean HbA2 is 4.8% with the range 3.7–7.0% [64].

This test works under the principle that HbF is resistant to alkaline treatment, while other hemoglobins are not [66]. Therefore, if hemoglobin solution of normal adult is mixed with alkaline solution, HbA, HbA2 is denatured, leaving only HbF dissolved in the solution. The dissolved HbF can be determined for its level by

The reagents that are required for this test comprise Drabkin's solution (0.20 g of K3Fe(CN)6, 0.05 g of KCN, DW to 1 L), 1.2 N NaOH, and saturated ammonium

To perform the test, 200 μL hemolysate is mixed in 3.8 mL of Drabkin's solution to prepare cyanmethemoglobin. Thereafter, 2.8 mL of cyanmethemoglobin solution is mixed with 200 μL of 1.2 N NaCl and shaked vigorously for 2 min exactly before adding 2.0 mL of saturated ammonium sulphate. Then, the precipitated hemoglobins are filtered out, and the OD540 of filtrate is measured and named OD540-filtrate. OD540 of total hemoglobin is measured in a mixture of 400 μL of hemolysate and

HbF or Alk F (%) = (OD540 − filtrate/ OD540 − total) × 10 (3)

Since HbF is determined by alkaline treatment, its level is then named Alkaline F or, simply, Alk F. Besides HbF, Hb Bart's (γ4) is also resistant to alkaline treatment. This protocol has maximum detection limit at only 50% of HbF. If the alkaline denaturation test is performed in fetal blood sample, the Alk F will not be more than 50%. Therefore, other techniques such as HPLC, CZE should be used to measure HbF in fetal blood. Alk F is not diagnostic marker for both β-thalassemia carriers and HbE carriers. However, increased HbF level presently is considered advantageous in

Normal range of HbA2 by this protocol is 1.3–3.7% (mean 2.5%) [64].

*4.1.2.3 Hemoglobin study by alkaline denaturation test of Betke*

6.75 mL of Drabkin's solution and named OD540-Total.

The percentage of HbF is calculated by the following Eq. 3:

If percent is less than 10, it is HbA2. If percent is 10 up, it is HbE. Normal range of HbA2 by this protocol is 2.62 ± 0.87% [52]. *Beta Thalassemia*

β+

β+

**Table 1.**

βO/β<sup>+</sup>

HbE/β<sup>+</sup>




/β+

*Hemoglobin pattern on CAE at pH 8.6 of β-thalassemia and HbE.*

HbE/βO-thalassemia (β<sup>E</sup>

using this technique, further tests must be done in order to determine the level of HbA2 and HbF. Raised level of HbA2 beyond normal range is the diagnostic marker for β-thalassemia carriers, while elevated level of HbF helps identify the high HbF condition found in the hereditary persistence of fetal hemoglobin (HPFH).

) A2FA

) A2FA

) EFA

/βO) EF

**Hb patterns on CAE at pH8.6 of adult**

This test is preliminarily aimed to quantify HbA2 levels that help diagnosis of the β-thalassemia carrier. However, HbE has the same pI as HbA2, thus these two hemoglobins are co-eluted. Microcolumn chromatography is an anion-exchange chromatography-based method. Anion-resin such as DEAE-cellulose or DEAE-Sephadex A50 suspended in appropriate buffer is packed in the microcolumn. On passing hemolysate through the packed resin, negatively charged hemoglobins binds to the resin at different binding affinity. HbA2 and HbE bind to the resin at the weakest strength (if there is no Hb Constant Spring) and are eluted out easily

There are two types of microcolumn chromatography, based on the anion-resin and buffers used. These include (1) DEAE-Sephadex A50 plus Tris-HCl-KCN buf-

DEAE-Sephadex A 50 resin suspended in Tris-HCl-KCN buffer (0.05 M Tris plus 0.1 g KCN/1 L, adjust pH with 4 N HCl) pH 8.5 is packed in pasture pipette to the height of 8–9 cm [52]. Then, the microcolumn is applied with 100 μL hemolysate, equilibrated with 10 mL THK buffer pH 8.5. Finally, 10 mL THK buffer pH 8.2 is applied to the microcolumn to elute HbA2 and/or HbE before 10 mL eluate is collected for measuring light absorbance (A) or optical density (OD) at 415 nm. This is then called A415-A2 or OD415-A2. For measuring OD415 of total hemoglobin or OD415-Total Hb, 100 μL hemolysate is mixed with 10 mL DW before measuring the absorbance. The level of HbA2 or HbE is calculated by the Eq. 1 shown below.

HbA2 or HbE (%) = [OD415 − A2/ OD415 − total Hb] × 100 (1)

*4.1.2 Hemoglobin study by microcolumn chromatography*

HbE heterozygote (carriers) EA

βO-thalassemia heterozygote (carriers) A2A

/β+


HbE homozygote EE (only 1 band of HbE)

with small amount of external anion such as CL<sup>−</sup>.

If percent is less than 10, it is HbA2. If percent is 10 up, it is HbE.

fer, and (2) DEAE-cellulose plus glycine-NaCl buffer.

*4.1.2.1 Microcolumn chromatography using DEAE-Sephadex A50 resin*

Normal range of HbA2 by this protocol is 2.62 ± 0.87% [52].

**134**

#### *4.1.2.2 Microcolumn chromatography using DEAE-cellulose*

This protocol was described by Wood [64] and modified in the author's laboratory in 2007 [65].

In modified protocol, DEAE-cellulose resin suspended in equilibrating buffer (0.2 M glycine + 0.01% KCN) is packed to the height of 2 cm in plastic microcolumn with 1.0-cm diameter. Then the microcolumn is applied by 50 μL hemolysate (prepared by mixing 1 part of PRC and 6 parts of 0.05% Triton X-100 as hemolysis buffer) and flushed with 5 mL eluting buffer (0.2 M glycine +0.01% KCN + 0.005 M NaCl). The 5-mL eluate is then collected and measured for absorbance or optical density at 415 nm (A415-A2 or OD415-A2). OD415 of total hemoglobin is measured in diluted hemolysate (50 μL hemolysate mixed with DW to 15 mL) and called OD415-Total.

Calculation of levels of HbA2 or HbE must follow the Eq. 2 shown below.

$$\text{HbA}\_2 \text{ or } \text{HbE (\%)} = \left[ \text{OD}\_{415} - \text{A}\_2 / \text{OD}\_{415} - \text{total} \times \text{3} \right] \times 100 \tag{2}$$

If percent is less than 10, it is HbA2.

If percent is 10 up, it is HbE.

Normal range of HbA2 by this protocol is 1.3–3.7% (mean 2.5%) [64].

For both protocols, if HbA2 level is less than 3.5%, the chance of β-thalassemia carrier is excluded. Instead, the cases may be either normal of α-thalassemia carriers. However, if HbA2 level is between 3.5 and 10.0%, the case is definitely β-thalassemia carrier. In β-thalassemia carriers, mean HbA2 is 4.8% with the range 3.7–7.0% [64].

#### *4.1.2.3 Hemoglobin study by alkaline denaturation test of Betke*

This test works under the principle that HbF is resistant to alkaline treatment, while other hemoglobins are not [66]. Therefore, if hemoglobin solution of normal adult is mixed with alkaline solution, HbA, HbA2 is denatured, leaving only HbF dissolved in the solution. The dissolved HbF can be determined for its level by measuring optical density at 540 nm.

The reagents that are required for this test comprise Drabkin's solution (0.20 g of K3Fe(CN)6, 0.05 g of KCN, DW to 1 L), 1.2 N NaOH, and saturated ammonium sulphate.

To perform the test, 200 μL hemolysate is mixed in 3.8 mL of Drabkin's solution to prepare cyanmethemoglobin. Thereafter, 2.8 mL of cyanmethemoglobin solution is mixed with 200 μL of 1.2 N NaCl and shaked vigorously for 2 min exactly before adding 2.0 mL of saturated ammonium sulphate. Then, the precipitated hemoglobins are filtered out, and the OD540 of filtrate is measured and named OD540-filtrate. OD540 of total hemoglobin is measured in a mixture of 400 μL of hemolysate and 6.75 mL of Drabkin's solution and named OD540-Total.

The percentage of HbF is calculated by the following Eq. 3:

$$\text{HbF or Alk F (9\%)} = \{\text{OD}\_{540} - \text{filterate} / \text{OD}\_{540} \text{ - total} \} \times 10 \tag{3}$$

Since HbF is determined by alkaline treatment, its level is then named Alkaline F or, simply, Alk F. Besides HbF, Hb Bart's (γ4) is also resistant to alkaline treatment. This protocol has maximum detection limit at only 50% of HbF. If the alkaline denaturation test is performed in fetal blood sample, the Alk F will not be more than 50%. Therefore, other techniques such as HPLC, CZE should be used to measure HbF in fetal blood.

Alk F is not diagnostic marker for both β-thalassemia carriers and HbE carriers. However, increased HbF level presently is considered advantageous in

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.
