**9. Identification of β-thalassemia and HbE by MAS-PCR**

This PCR technique was established by author and named multiplex allele specific (MAS)-PCR [29]*.* MAS was intended to be used for identifying βThalassemia and βE -mutations in samples that have already been diagnosed to be β-thalassemia carrier, HbE carrier, and SEA-α thalassemia 1 carrier. Therefore, only mutant primers are put together in this MAS-PCR. However, the internal control of this PCR protocol is the amplified products generated by the normal pair of primers for α-globin gene cluster.

**Procedure**: The PCR was performed in a total volume of 25 μL containing 1.1–1.4 ng genomic DNA, 140 μM dNTPs, 0.1 unit/μL of DNA polymerase, 0.25 ng/ μL "Beta-common-multiplex" primer (5′-AAG AGC CAA GGA CAG GTA CGG CTG T-3′), 0.125 ng/μL "Beta-17-multiplex" primer (5′-CCA ACT TCA TCC ACG TTC ACG TA-3′), 0.125 ng/μL "Beta cds-41/42-multiplex" primer (5′-AGA TCC CCA AAG GAC TCA ACC T-3′), 0.125 ng/μL "Beta-E-multiplex" primer (5′-CGT ACC AAC CTG CCC AGG GCC AT-3′), 25 ng/μL "SEA-1-multiplex" primer (5′- TGA CTC CAA TAA ATG GAT GAG GA-3′), 0.25 ng/μL "SEA-2-multiplex" primer (5′-GCC TGC GCC GGG GAA CGT AAC CA-3′)and 0.5 ng/μL "SEA-3-multiplex" primer (5′-CGC CAA AGA TGG CTA CTC GGA GA-3′) in 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 2.0% DMSO and 2.0 mM MgCl2.

**Thermal cycles**: A total of 37 thermal cycles were carried out with each cycle comprising denaturation at 95°C for 1 min, primer annealing at 62°C for 30 s, and primer extension at 72°C for 30 s. Initial denaturation was extended to 5 min and final extension was prolonged to 7 min.

**Detection of amplified products**: The PCR products were separated via 2.0% agarose gel electrophoresis and visualized by the UV-transilluminator (**Figure 28**).

#### **Figure 28.**

*MAS-PCR for identifying β-thalassemia mutation, β<sup>E</sup> -mutation, and SEA-α thalassemia 1 mutation. The 653-bp amplified products are the internal control generated by the α-globin gene cluster specific oligonucleotide primers. Lanes 1–5 are positive for β<sup>E</sup> mutation as the 293-bp amplified products are seen. Lane 6 is positive for both SEA-α thalassemia 1 and β<sup>E</sup> mutations as both 753-bp amplified products and 293-bp amplified products are seen. Lane 7 is positive for both SEA-α thalassemia 1 and β17 mutation as both 753-bp amplified products and 268-bp amplified products are seen. Finally, lane 8 is positive for SEA-α thalassemia 1 and β41/42 mutation as both 753-bp amplified products and 466-bp amplified products are seen (modified from [82]).*

**153**

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

steps of laboratory diagnosis.

negative results and *vice versa.*

bp, and 268-bp products indicates presence of β41/42, β<sup>E</sup>

**10. Pitfalls in laboratory diagnosis of β-thalassemia and HbE**

results will be obtained if NaCl concentration is too high, and *vice versa.*

**10.1 Pitfalls in screening tests for β-thalassemia and HbE carriers**

screening for β-thalassemia and HbE carriers.

**10.2 Pitfalls in confirmatory tests for β-thalassemia and HbE**

**Interpretation**: All reactions must have the 653-bp control products. Presence of 753 bp product indicates presence of the SEA-α thalassemia 1. Presence of the 466-bp, 293-

There are several pitfalls to be concerned when using laboratory data in diagnosis of β-thalassemia and HbE. The pitfalls are in both screening and confirmatory

HbE tube test relies on concentration of NaCl. Therefore, exact amount of NaCl in the reagent must be prepared following the suggested ingredient. False positive

HbE screen test relies on pH-based microcolumn chromatography. Therefore, exact pH of buffer in the test kit must also be prepared. Too high pH causes falsely

1.**Pitfalls in one-tube osmotic fragility test (OFT).** The concentration of reagent must be exactly 0.36 and 0.45%, otherwise false positive results will be obtained if concentration is over 0.36 or over 0.45%. In contrast, false negative results will be obtained if concentration is less than 0.36 or 0.45%. Anemic blood samples will also give positive results. Therefore, Hb/Hct must also be checked if the results are positive. Not all HbE carriers have positive OFT results. Therefore, blood samples having negative OFT results must also be sent for HbE screen.

2.**Pitfalls in MCV and MCH evaluation.** MCV/MCH are the numeric data that must be obtained from automated blood cell counters that have good quality control. MCV must be obtained using fresh blood, but MCH may be obtained using 1 week-old blood. MCV is directly measured in automated blood counter, while MCH is generated by calculation. Thus, if blood samples have high degree of variation of red blood cell sizes or anisocytosis, false MCV values may be obtained. This situation may be found in β-thalassemia carriers or HbE carriers with co-existence iron deficiency anemia during treatment. Not all HbE carriers have MCV/MCH values below cutoff points. Therefore, all blood samples sent for MCV/MCH determination must also be sent for HbE screen. Most importantly, some blood samples may have discordant MCV-OFT results. It is then highly recommended to perform both OFT and MCV/MCH for

3.**Pitfalls in HbE screening test.** DCIP test is based on using the oxidizing reagent; dichlorophenolindophenol. This reagent can be reduced over long storage and the screening results will be falsely negative. Therefore, if color of the DCIP reagent turns deeply blue, it should not be used. HbH also denatured in the DCIP reagent. Therefore, positive samples must be checked if they are HbH disease.

Normal hemoglobin type depends on age of the patients. Thus, reading and interpreting the hemoglobin typing results by all methodologies, age of the patients

must be taken into account. In addition, transfused blood interferes the real

, and β

17, respectively (**Figure 28**).

*Beta Thalassemia*

genotype β<sup>E</sup>

βE

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

/β<sup>E</sup>

samples are negative for the β<sup>E</sup>

**Interpretation**: Both M and N-reactions must have the 314-bp control products and the results can be read. If both M- and N-reactions have 260-bp PCR products,

represents HbA; T represents other types of β-globin gene mutation). If the 260-bp PCR products are seen in only M-reaction, the samples are homozygote for β<sup>E</sup>

This PCR technique was established by author and named multiplex allele specific (MAS)-PCR [29]*.* MAS was intended to be used for identifying βThalassemia and

**Thermal cycles**: A total of 37 thermal cycles were carried out with each cycle comprising denaturation at 95°C for 1 min, primer annealing at 62°C for 30 s, and primer extension at 72°C for 30 s. Initial denaturation was extended to 5 min and

**Detection of amplified products**: The PCR products were separated via 2.0% agarose gel electrophoresis and visualized by the UV-transilluminator

*653-bp amplified products are the internal control generated by the α-globin gene cluster specific oligonucleotide* 

*are seen. Lane 7 is positive for both SEA-α thalassemia 1 and β17 mutation as both 753-bp amplified products and 268-bp amplified products are seen. Finally, lane 8 is positive for SEA-α thalassemia 1 and β41/42 mutation* 

*as both 753-bp amplified products and 466-bp amplified products are seen (modified from [82]).*

*-mutation, and SEA-α thalassemia 1 mutation. The* 

 *mutation as the 293-bp amplified products are seen. Lane 6 is positive for* 

 *mutations as both 753-bp amplified products and 293-bp amplified products* 


HbA; T represents other types of β-globin gene mutation) (**Figure 27**).

**9. Identification of β-thalassemia and HbE by MAS-PCR**

(pH 8.8), 50 mM KCl, 2.0% DMSO and 2.0 mM MgCl2.

final extension was prolonged to 7 min.

*MAS-PCR for identifying β-thalassemia mutation, β<sup>E</sup>*

*primers. Lanes 1–5 are positive for β<sup>E</sup>*

*both SEA-α thalassemia 1 and β<sup>E</sup>*

(**Figure 28**).

with genotype of either β<sup>E</sup>

. If samples have the 260-bp PCR products in only N-reaction, the

with genotypes of either βA/βA or β<sup>T</sup>

/βA or β<sup>E</sup>

/β<sup>T</sup>

/β<sup>T</sup> (A

with

(A represents

**152**

**Figure 28.**

**Interpretation**: All reactions must have the 653-bp control products. Presence of 753 bp product indicates presence of the SEA-α thalassemia 1. Presence of the 466-bp, 293 bp, and 268-bp products indicates presence of β41/42, β<sup>E</sup> , and β 17, respectively (**Figure 28**).
