\r\n\tThis book is intended to provide a series of peer reviewed chapters that the guest editor believe will aid in increasing the quality of the research focus across the growing field of grain and seeds compound functionality research. Overall, the objective of this project is to serve as a reference book and as an excellent resource for students, researchers, and scientists interested and working in different functional aspects of grain and seed compounds, and particularly for the scientific community to encourage it to continue publishing their research findings on grain and seed and to provide basis for new research, and the area of sustainable crop production. \r\n\t
",isbn:"978-1-83968-591-0",printIsbn:"978-1-83968-590-3",pdfIsbn:"978-1-83968-592-7",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"9268519d1e294c5edf8e964a122e4c91",bookSignature:"Dr. Jose Carlos Jimenez-Lopez",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/9699.jpg",keywords:"Dormancy, Germination, Development, Storage Protein, Seed Protein, Seed Storage Protein Gene, Nutritional Composition, Pharmaceutical Applications, Antioxidants, Molecular Allergy, Food Allergy, Diagnosis and Therapy",numberOfDownloads:240,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"August 26th 2020",dateEndSecondStepPublish:"September 23rd 2020",dateEndThirdStepPublish:"November 22nd 2020",dateEndFourthStepPublish:"February 10th 2021",dateEndFifthStepPublish:"April 11th 2021",remainingDaysToSecondStep:"7 months",secondStepPassed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:"Dr. Jimenez-Lopez is an active member of the Spanish Legume Society, International Legume Society, Spanish Society for Microscopy, and European Microscopy Society. He is the editor of multiple books and serves as the peer reviewer for journals published by Elsevier, Springer, Wiley, Frontiers, etc. In 2012 he was awarded by Marie Curie Research Fellowship.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"33993",title:"Dr.",name:"Jose Carlos",middleName:null,surname:"Jimenez-Lopez",slug:"jose-carlos-jimenez-lopez",fullName:"Jose Carlos Jimenez-Lopez",profilePictureURL:"https://mts.intechopen.com/storage/users/33993/images/system/33993.jpg",biography:"Dr. Jose Carlos Jimenez-Lopez, has studied Biochemistry and Molecular Biology (1998) and obtained Bs. in Biological Sciences (2001), Ms. in Agricultural Sciences (2004), University of Granada, Spain and PhD degree in Plant Cell Biology (2008) at the Spanish National Research Council (CSIC). He was a Full-time Postdoctoral research associate at Purdue University, USA (2008-2011). Marie Curie Research Fellow (FP7-PEOPLE- 2011-IOF) (2012-2015) at the University of Western Australia and CSIC working in human health benefits of legume seed proteins, their allergy molecular aspects and cross allergenicity. He is a Senior Research Fellow (Ramon y Cajal research program - MINECO, 2016 - present), currently working in the functionality, health benefits, and allergy implications of proteins from reproductive tissues (pollen and seeds) in crop species of agro-industrial interest (mainly legumes). He is an Author of more than 60 peer-review journal articles, 25 book chapters. His work has been presented in more than 130 international congresses. He is an Active member of different Scientific Societies: Spanish and International Legume Society; Spanish and EU Microscopy societies. He is Editor of multiple books, Reviewer for more than 35 Peer-Review Journals of editorial as Elsevier, Springer, Wiley, Frontiers, etc, and international expert panels member for funding grants evaluation.",institutionString:"Spanish National Research Council (CSIC)",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"6",totalChapterViews:"0",totalEditedBooks:"6",institution:{name:"Spanish National Research Council",institutionURL:null,country:{name:"Spain"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"5",title:"Agricultural and Biological Sciences",slug:"agricultural-and-biological-sciences"}],chapters:[{id:"74440",title:"Pea Seed Proteins: A Nutritional and Nutraceutical Update",slug:"pea-seed-proteins-a-nutritional-and-nutraceutical-update",totalDownloads:60,totalCrossrefCites:0,authors:[{id:"311935",title:"Dr.",name:"Prashant",surname:"Kaushik",slug:"prashant-kaushik",fullName:"Prashant Kaushik"}]},{id:"75744",title:"Health Benefits and Industrial Applications of Functional Cowpea Seed Proteins",slug:"health-benefits-and-industrial-applications-of-functional-cowpea-seed-proteins",totalDownloads:39,totalCrossrefCites:0,authors:[null]},{id:"75313",title:"RNAi-Mutants of Sorghum bicolor (L.) 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From chapter submission and review, to approval and revision, copy-editing and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. 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1. Introduction
Approximately one in four prescription drugs from pharmacies in the U.S., Canada, and Western Europe have active ingredients that are plant derived (Balick and Cox, 1997). Edible and even non-edible plants have long been considered sources of anticancer drugs. Indeed, our laboratory (Dr. Booth) published a paper two years ago (Capua et al., 2010) showing that even non-edible and non-tropical desert plants have bioactivity against a wide variety of human cancer cells. From the literature, it is clear that diets rich in grains, fruits, and vegetables are known to reduce cancer risk (Ferguson, et al., 2004; Guthrie and Carrol, 1998) especially those rich in antioxidant activity.
Over the last 20 years, a number of juices and nutritional liquid supplements have appeared on the market purporting high amounts of antioxidant activity and suggesting a number of benefits to human health. In the corridor of Utah Valley, for example, there are at least a dozen manufacturing centers for plant-derived supplements within 50 miles of each other. While these supplements have caught the interest of health enthusiasts throughout the world, it is of interest that very few toxicology studies have been published on these supplements especially with reference to anticancer dose-response curves and antioxidant activity. Three years ago, our laboratory was asked to evaluate the potential health effects of a new nutritional supplement referred to as Galaxy®. This product was of interest to us because it contained a variety of bioactive ingredients including several superfruits (a marketing term for fruits with unusual nutrient and antioxidant properties).
We agreed to investigate this product on five conditions:
We develop our own research protocols.
Principal investigators accept no personal compensation for the studies.
We were allowed to present our data at professional conferences and symposia.
We are allowed to publish our results (regardless of the outcome) in the peer-reviewed literature.
They provide the product to our lab free of charge.
The company agreed without hesitation which was a fresh departure from the traditional position of the industry that generally requires non-disclosure documents, no-publication policies, and restricted professional presentations of the data sets. Thus, with that agreement, we pressed forward with the following objectives:
Investigate the antioxidant activity of the freeze-dried product.
Develop dose-response curves using Galaxy® on a variety of human cancer cell lines including calculation of EC50s.
Determine if a correlation exists between anticancer activity and antioxidant activity using Galaxy® and several selected superfruits.
Compare the toxicity of an approved FDA drug (paclitaxel) with the particulate (most active) fraction of Galaxy®.
Develop Bioactivity Indices for Galaxy® and selected superfruits.
Develop Selectivity Indices which compares the cytotoxicity between cancer cells and normal cells.
Show the effect of Galaxy® on blood glucose levels of senior athletes (n = 308).
Demonstrate the effect of Galaxy® on the white blood cell (WBC) count of an acute lymphocytic leukemia (ALL) patient over a number of months without chemotherapy or any other treatment intervention.
Determine the simple carbohydrate and amino acid content of a freeze-dried sample of Galaxy®.
2. Materials and methods
Galaxy, a nutritional supplement, was provided by JoyLife International. This blend contains 32 bioactive ingredients. Bioassay data from this product were collected using the straight sample (from the container), as a freeze-dried sample (Fig. 1), supernatant, or particulate fraction. Bioassay procedures for all these matrices were completed using methods developed previously from Brigham Young University (BYU) laboratories (Capua et al., 2010).
All cell lines were grown in the laboratories of BYU or Reaction Biology Corporation. The purity of the cell lines was checked periodically using an inverted microscope (Fig. 2).
The ORAC assay was performed according to published protocols (Parker et al., 2007; Fig. 3). Preparation of the freeze-dried Galaxy sample for sugar extraction was completed by extracting 5 g of the sample with 50 mL of 70 % (w/v) methanol solution in a 100 mL Ehrlenmeyer Flask for 24 hours using a stirring bar. The extract was then analyzed for sugars and amino acids standardized GC/MS procedures from protocols developed at the BYU College of Life Sciences Chromatography Facility.
Figure 1.
Dr. Gary M. Booth (left) and Kyle Lorenzen preparing freeze-dried samples of Galaxy®.
Figure 2.
Dr. Gary M. Booth (standing) and Matt Dungan viewing the effect of Galaxy® on cancer cells in an inverted microscope.
Figure 3.
Dr. Tory L. Parker preparing Galaxy® samples for determination of oxygen radical absorption capacity (ORAC).
Figure 4.
Collection of blood glucose samples from the athletes (n = 308) of the 2011 Huntsman Senior World Games for investigation of the effect of Galaxy® on blood glucose.
Glucose measurements were determined using a Bayer Contour Glucosometer on 308 senior athletes during the 2011 Huntsman Senior Games held in St. George, Utah. The average age of the participants was 64.5 ± 4 years (Fig 4).
Onxol® (paclitaxel; also called Taxol®) an FDA‐approved drug for breast cancer was purchased from Cancer Care Northwest in Spokane, WA. This drug was used to compare with the most active fraction (particulate) of Galaxy®.
3. Results and discussion
It is now well documented through epidemiological studies that diets rich in fruits and vegetables can reduce cancer and other chronic diseases (Boivin et al., 2007; Block et al., 1992; Steinmetz et al., 1991). It is believed that the reduction in these chronic diseases is related to the diversity and high concentrations of antioxidants, known collectively as phytochemicals. Furthermore, it has been generally documented that the complex mixtures of phytochemicals in fruits and vegetables are more effective than their individual components in preventing cancer through a variety of mechanisms that include both additive and synergistic effects (Liu et al., 2004; Seeram et al., 2005; Mertens-Talcon et al., 2003; Zhou et al., 2003).
In the U.S., a recent survey (Yang et al., 2011) showed that the total antioxidant capacity (TAC) was positively correlated with daily consumption of fruits and fruit juices, vegetables, and antioxidant-containing beverages. The major sources of the dietary TAC in the U.S. were teas, dietary supplements, and fruits and fruit juices which accounted for 28%, 25%, and 17% of the TAC respectively. Unfortunately, vegetables only contributed 2% of the dietary TAC. Recognizing that their diets may not be optimum, many people supplement their diet with powdered or liquid supplements, such as Galaxy® to get their “daily dose” of dietary TAC.
The nutritional supplement/juice industry has shown consistent growth over the last several years, mostly through consumption by the older generation. However, even though this economic growth spurt has stimulated the economy and possibly even contributed to the health of our population, the supporting scientific data from these products has often been lacking. The experimental data from this product now follows:
A freeze dried sample of Galaxy® had an ORAC value of 178.10 ± 15.02 µmoles TE/g (n=4), while the fresh product was 35.6 ± 3.1 µmoles TE/g wet weight. The USDA recommends (USDA, 1999) that each person consumes approximately 3000-5000 ORAC units per day. As of this writing, the average person in the U.S. consumes less than 1000 ORAC units/day. Based on our data (200 mg particulates/one mL Galaxy® ), if a person consumes 30 mL of Galaxy® per day, they would consume about 1068 ORAC units per day or 21-36% of the recommended units per day. And if one consumes it 2X/day, the values double, or 42-72% of the recommended units. Thus, one bottle of Galaxy® contains about 26,715 ORAC units using the freeze-dried data or 26,700 ORAC units if the fresh weight data are used. Thus, Galaxy® juice from the bottle has a higher ORAC value than 21 of the 22 fruit juices listed by the USDA (Haytowitz and Bhagwat, 2010). Only black raspberry juice (ORAC = 10,460 µmoles TE/100 g wet weight) had a higher antioxidant rating.
The freeze-dried Galaxy® ORAC value of 178.1 µmoles TE/g is approximately the same antioxidant capacity as that reported for cranberry (Sun et al., 2002) which is considered the reference standard for calculation of Biological Indices (BI). Thus, Galaxy® has a BI of 3.40 which of course is 3.40X higher than the cranberry standard (BI = 1). Table 1 shows a summary of the ORAC values, EC50s, and BI for Galaxy® and several superfruits of which three (mangosteen, GAC, and Acai) are contained in this product. The BI is a useful parameter to compare the combined effects of anticancer activity and antioxidant activity. Any BI above 1 is considered relatively good. Thus, Galaxy has a good BI compared against the individual fruits. Except for mangosteen and GAC, all of these superfruits were in the same order of magnitude ranging from 1.73 to 8.46. There was no correlation (r2 = 0.07; p = 0.473) between the anticancer (EC50) parameter and the ORAC value for several superfruits suggesting that antioxidative activity may not be totally related to in vitro anticancer activity (Fig. 5). Still, a careful look at Fig. 5 shows that five of the nine samples (including Galaxy® particulates) had ORAC values above 150 µmoles TE/g and an EC50 less than 1mg/mL. Thus, ORAC and EC50s values for these superfruits appear to be related, just not in a clear inverse linear fashion.
Figure 5.
Plot of the relationship between ORAC values and EC50 values for Galaxy® and eight superfoods
Correlation studies on these parameters with other fruits have also been equivocal; some are positively correlated (Wang and Lewers, 2007; Faria et al., 2006) while others are not (Boivin et al., 2007). A recent review (Wang et al., 2011) demonstrated that there is no conclusive proof that high antioxidant activity is a good indicator of high anticancer activity but left the reader with the challenge to continue to test this hypothesis until it is unequivocally resolved one way or the other. Similar studies with total phenolics have also shown mixed results. Some studies of total phenolics contained in traditional fruit, have shown to be highly correlated with anticancer activity (Silva et al., 2006; Atmani et al., 2011) and they (phenolics) are also highly correlated with ORAC values (Atmani et al., 2011; Kalt et al., 2003). However, some researchers have shown that there is no correlation between total phenolics and anticancer activity (Thompson et al., 2009; Sun et al., 2002; Weber et al., 2001). Thus, the expected link between ORAC values and total phenolics and anticancer activity continues to be a subject of debate in the literature.
Fruit
ORACa
Scoreb
EC50 (mg/mL)
Scorec
BId
Mangosteen
251.60
1.42
0.30
48.33
24.88
GAC
153.00
0.86
0.30
48.33
24.60
Acai
425.10
2.42
1.00
14.50
8.46
Goji
378.10
2.14
1.10
13.20
7.67
Galaxy®
178.10
1.00
2.30
6.30
3.65
Sea Buckthorn
377.10
2.13
4.10
3.54
2.84
Noni
151.80
0.86
5.60
2.59
1.73
Cranberry
354.27
1.00
14.50
1.00
1.00
Table 1.
Summary of the total antioxidant activity (ORAC), antiproliferative (EC50), and the ranked Bioactivity Index (BI) for Galaxy® and seven superfruits; mangosteen, acai, goji, and cranberry are found in Galaxy found in Galaxy® compared against the cranberry standard.
Fig. 6 shows the in vitro effect of Galaxy® straight (raw product) on MDA-MB-231 breast cancer cells. Galaxy® has an EC50 = 2.3 which is an order of magnitude lower (more cytotoxic) than most traditional fruits (Weber et al., 2001) but higher (less cytotoxic) than most chemotherapy drugs (Frankfurt and Krishan, 2003).
Figs. 7 and 8 show the in vitro dose-response curve for the effect of the Galaxy® supernatant and particulate fractions respectively on breast cancer cell line MDA-MB-231. It is clear that the supernatant (Fig. 7: EC50=3.4) and the particulate fraction (Fig. 8: EC50=0.075 mg/mL) were both cytotoxic to this cell line, but the particulate fraction was 47X more toxic than the supernatant fraction. This indicates that most of the phytochemicals (98%) in Galaxy® contributing to the anticancer activity are membrane-bound. These data are in contrast to those reported by Wang et al. (1996) who found less than 10% of the ORAC activity in a wide variety of different fruit pulp or particulates; they found most of the bioactivity in the extracted juice. However, the USDA ORAC database (Haytowitz and Bhagwhat, 2010) apparently also showed that most of the antioxidant activity in a number of fruits was associated with the pulp, and not the juice.
Figure 6.
Galaxy® (straight from container) effect on MDA-MB-231 breast cancer Cells (EC50= 2.3 mg/mL).
When the particulate dose-response curve was compared to the FDA-approved breast cancer drug Onxol® (paclitaxel), the two curves tended to track each other with both EC50s being in the same order of magnitude (Fig. 8; EC50=0.03 mg/mL for paclitaxel; EC50=0.075 mg/mL for the Galaxy® particulates). The EC50 for paclitaxel in our study was in the same order of magnitude as that reported by Danhier et al. (2009) for HeLa cells, but higher than that reported by Yu et al. (2004) for MCF-7 and MDA-MB-231 cell lines.
Figure 7.
Galaxy® (supernatant) toxicity to MDA-MB-231 Human Breast Cancer Cells (EC50 = 3.4).
Figure 8.
Comparison of Galaxy® Particulates and Onxol® (FDA approved drug) against Human Breast Cancer Cell Line MDA-MB-231 (Galaxy® EC50 = 0.075 mg/mL; Onxol® EC50 = 0.03 mg/mL).
We have also tested Galaxy® against several other human cancer cell lines and calculated a Selectivity Index (SI) which compares EC50s of cancer cells and normal cells. Table 2 summarizes the EC50s and ranked (SI) for various samples of Galaxy® on breast cancer, lung cancer, and liver cancer compared to its effect on normal tissues. The higher the SI values, the higher the selectivity of Galaxy® against cancer cells compared to the normal tissues. Any SI above 2 is considered a reasonably selective SI (Basida et al., 2009). Note that the SI values for Galaxy® ranged from 3.2 (for lung cancer) to over 370 for the particulate fraction. Badisa et al. (2009) reported that pure anticancer compounds (piperidinyl-DES, Pyrrolidinyl-DES, and 4-hyroxy tamoxifen) had SI values that ranged from 1.29 to >2.84. They further indicated that an SI value less than 2 probably indicates general toxicity of pure compounds (Koch et al., 2005). Thus, one of their three compounds, pyrrolidinyl-ES showed a high degree of cytotoxic selectivity, while 4-hydroxy tamoxifen (an anti-breast-cancer drug) showed an SI of less than 2 suggesting general toxicity to cells according to the recommendation of Koch et al.(2005).
Cell Line
Cell Type
Galaxy Sample
EC50 (mg/mL)
SI*
MDA-MB-231
Breast cancer
Particulates
0.075
371
Breast cancer
Unfractionated
2.30
12
Breast cancer
Supernatant
3.5
8
Hs578Bst
Normal breast cells
Freeze-dried
27.80
-
HepG2
Liver cancer
Freeze-dried
3.44
3.5
THLE-3
Normal hepatocytes
Freeze-dried
11.99
-
A-549
Lung cancer
Freeze-dried
6.20
3.20
MRC-5
Normal lung cells
Freeze-dried
19.68
-
Table 2.
Summary of the EC50s and ranked Selectivity Indices (SI) for Galaxy® samples on human breast cancer, lung cancer, and liver cancer compared to normal human tissues.
In addition, Al-Qubaisi et al. (2011) reported an SI of 7.6 for liver cell comparisons using Goniothalamin isolated from a plant compared to Galaxy®’s SI of 3.5; however, Galaxy® was over 1,700X less toxic to normal liver cells when the EC50s of Galaxy® and Goniolthalamin were compared. Apparently, phytotherapy (using mixtures of dozens of fruit-based and vegetable-based polyphenols and other antioxidants such as those found in Galaxy®) are often less toxic than the purified compounds used in mainstream cancer therapy. Indeed, Krishna et al. (2009) has suggested that no anti-neoplastic drug is devoid of side effects which includes the widely used anti-cancer drug, paclitaxel. Our in vitro data suggests that the phytochemicals found in Galaxy® are quite selective for cancer cells. While these preliminary data on Galaxy® are encouraging, the authors caution all readers to not extrapolate conclusions from the in vitro data to what might happen in large randomized double-blind in vivo investigations.
Figs. 9-12 summarizes the dose-responses curves of freeze-dried Galaxy® on colon cancer, prostate cancer, lung cancer, and liver cancer. The EC50s ranged from about 4.0-11 mg/mL which is an order of magnitude higher in cytotoxicity (lower EC50) than that reported for four raspberry varieties and one apple variety (Weber et al., 2001) and for a variety of other fruits (Sun et al., 2002). Thus, Galaxy® seems to have a broad-spectrum of in vitro antiproliferative and antioxidant activities. However, it is still not known if or how the individual components in the product interact to produce these toxicities.
Three hundred and eight senior athletes had their baseline blood glucose levels taken, then drank 30 mL of Galaxy®, and then waited about one hour and had their blood glucose taken again (post-treatment). The data show that Galaxy® apparently does not spike blood glucose (Table 3) which is probably a result of the balance of simple sugars found in the product (Fig. 13) which was 16.5% xylitol; 42.2% fructose; and 41.3% glucose. Galaxy® also contains nine essential amino acids.
Figure 9.
Effect of Freeze-dried Galaxy® on Human Colon Cancer Cells (EC50 = 10.5 mg/mL).
Figure 10.
Effect of Freeze-dried Galaxy® on DU 145 Human Prostate Cancer Cells (EC50 = 5.3 mg/mL).
Figure 11.
Effect of Freeze-dried Galaxy® on A-549 Human Lung Cancer Cells (EC50 = 5.0 mg/mL).
Figure 12.
Effect of Freeze-dried Galaxy® on HepG2 Human Liver Cancer Cells (EC50 = 5.0 mg/mL).
Figure 13.
Chromatogram of the types of natural sugar in Galaxy®.
JoyLife International does not make health claims for their product because it is marketed as a nutritional supplement, not as a medicine. However, we do have one data set on a 33 year old Caucasian female who suffers from Acute Lymphocytic Leukemia (ALL) who agreed to take the product for seven months without any other medical intervention, e.g. no chemotherapy. This was done under the strict supervision of her health-care providers. Fig. 14 shows that her white blood cell count dropped from 68,000 to 21,700 cells/µL, coupled with a substantial improvement of her secondary health parameters associated with the disease (e.g., lethargy, bruising, muscular weakness, etc). This resulted in a 68% drop in the WBC over the seven month treatment period. The regression (r2 = 0.77) relationship between the seven-month Galaxy® treatment and changes in WBC suggests that 77% of the variability in the changes in the slope of the graph was likely due to the Galaxy® treatment. The authors are not drawing any conclusions from this single data set, nor should the reader, but it is provided since it was done under the close scrutiny and supervision of her oncologists and without any other intervention. This study is on-going.
Figure 14.
Effect of Galaxy® on WBC Count in an Acute lymphocytic Leukemia Patient
4. Summary and conclusions
Galaxy® is a nutritional food blend that contains 32 bioactive components including thirteen high antioxidant fruits. A freeze-dried sample of this blend has an ORAC score of 178.10 ± 3.1 µmoles TE/ g of freeze-dried product or 35.6 µmoles TE/g fresh product. Based on a dose of 30 mL/day, a person would consume 1068 ORAC units/dose. This value represents 21-36 % of the daily recommended ORAC units (3000-5000 ORAC units/day) suggested by the USDA. Thus, the entire bottle of Galaxy® contains about 26,700 ORAC units. It is likely that the antioxidants contained in this product contributed to the in vitro anticancer activity. These anticancer EC50s ranged from 0.075 to 11 mg/mL on breast, colon, prostate, lung, and liver cancers. The raw product, the supernatant, and particulate fractions of Galaxy® were all bioactive, although the particulate fraction was over 40X more active than either the raw product or supernatant suggesting that about 98% of the bioactivity is contained in the particulate fraction. In fact, the particulate fraction dose-response curve tracked the dose-response curve of Onxol® (an FDA-approved drug for breast cancer) with EC50s (EC50 = 0.075 mg/mL for Galaxy® particulates and EC50 = 0.03 mg/mL for Onxol®) that were in the same order of magnitude of each other.
There was a no correlation between the anticancer activity (EC50s) and the ORAC values of the 9 superfruits investigated (four of which are found in the product) (r2= 0.0758; p = 0.473), although four of the nine superfruits (including Galaxy®) had an EC50 < 1.0 and ORAC values greater than 150 umoles TE/g. From these data, it appears that ORAC units may somehow still be connected, though it is apparently not a clear inverse relationship. Galaxy® was also shown to have a Bioactivity Index (BI) of 3.40, which was 3.40X more active than the cranberry BI reported in the literature.
Galaxy® additionally did not spike blood glucose levels of 308 participants during the 1 ½ hours following the normal dose of 30 mL (n=308). This was likely due to the balance of simple sugars found in the product: 16.5% xylitol; 42.2% fructose; and 41.3% glucose (Fig. 13). In addition, Galaxy® contains nine essential amino acids.
JoyLife International makes no health claims for this product because it is marketed as a nutritional supplement, not a medicine; however, in cooperation with her physician, one patient with ALL showed a 68% drop in WBC count (from 68,000/uL to 21,700/uL) after seven months post-treatment with Galaxy® (30 mL/day) without any other medical interventions including chemotherapy. In addition, there were significant qualitative improvements in the patient’s secondary health parameters. This study is on-going.
Research has clearly shown that people in the U.S. are not eating enough vegetables, since only 2 % of TAC consumed in any form comes from vegetables.
These data have suggested other experiments that need to be considered. For example, investigations are needed to determine how these cancer cells are dying, i.e., by apoptosis or necrosis as well as the toxicological contribution of each of the individual components of the blend. The issue of possible synergistic activity among the ingredients also needs further study. The effect of Galaxy® on rodent models with induced inflammatory diseases need to also be considered. And finally, larger randomized double-blind experiments are needed to validate the data from the ALL patient and from other people suffering from chronic diseases.
Acknowledgement
The authors are not associated with the sale, marketing, or distribution of Galaxy®. All of the data were generated from protocols developed by the principal investigators without contact with the company. All of the data contained in this manuscript has not been submitted to any other publisher.
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Materials and methods",level:"1"},{id:"sec_3",title:"3. Results and discussion",level:"1"},{id:"sec_4",title:"4. Summary and conclusions",level:"1"},{id:"sec_5",title:"Acknowledgement",level:"1"}],chapterReferences:[{id:"B1",body:'Al-QubaisiM.RozitaR.-KS.Yeap-RA.Omar-MA.AliAlitheenN. B.2011Selective cytotoxicity of Goniothalamin against hepatoblastoma HepG2 cells. Molecules 1629442959'},{id:"B2",body:'AtmaniDina.BegnaM.Ruiz-LarreaJ. I.Ruiz-SanzL. J.LizcanoF.BakkaliDjebbarAtmani.2011Antioxidant potential, cytotoxic activity and phenolic content of Clematis flammula leaf extracts. J. Med. Pl. Res. 54589598'},{id:"B3",body:'BadisaR. B.Darling-ReedS. F.JosephP.CooperwoodJ. S.LatinwoL. M.GoodmanC. B.2009Selective cytotoxic activities of two novel synthetic drugs on human breast carcinoma MCF-7 cells. Anticancer Res. 2929932996'},{id:"B4",body:'BalickMichael.and Paul Alan Cox. 1996Plants, People, and Culture. 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E.DonnerH.ForneyC. F.2003Oxygen radical absorbing capacity, anthocynanin and phenolic content of highbush blueberries (Vaccininium corymbosum L.) during ripening and storage. J. Amer. Hort. Sci. 1286917923'},{id:"B15",body:'KochA.TamezP.PezzutoJ.SoejartoD.2005Evaluation of plant used for antimalarial treatment by the Massai of Kenya. J. Ethnopharmacol. 1019599'},{id:"B16",body:'KrishnaI. V.VnajaG. R.KumarN. S.SumanG.2009Cytotoxic studies of anti-neoplastic drugs on human lymphocytes-in vitro studies. Cancer Biomark. 56261262'},{id:"B17",body:'LiuR. H.2004Potential synergy of phytochemicals in cancer prevention: mechanism of action. J. Nutr. 134:3479S EOF3485S EOF'},{id:"B18",body:'Mertens-TalcottS. U.TalcottS. T.PercivalS. S.2003Low concentrations of quercetin and ellagic acid synergistically influence proliferation, cytotoxicity and apoptosis in MOLT-4 human leukemia cells. J. Nutr. 13326692674'},{id:"B19",body:'ParkerT. L.-HX.WangJ.PaziminoEngesethN. 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Fd Chem. 5074497454'},{id:"B24",body:'ThompsonM. D.2009Chemical composition and mammary cancer inhibitory activity of dry bean. Crop Sci. 491179186'},{id:"B25",body:'USDA Human Research Center on Aging.Feb. 1999Can foods forestall aging?” Agri. Res. Feb. 1999.'},{id:"B26",body:'WangHong.GuohuaCao.RonaldL.Prior1996Total antioxidant capacity of fruits. J. Agric. Fd Chem. 443701705'},{id:"B27",body:'WangS.MecklingK. A.MarconeM. F.KakudaY.TsaoR.2011Can phytochemical antioxidant rich foods act as anti-cancer agents? Food Res. Intern. 4425452554'},{id:"B28",body:'WangS. Y.LewersK. S.2007Antioxidant activities and anticancer cell proliferation properties of wild strawberries. J. Amer. Soc. Hort. Sci. 132647658'},{id:"B29",body:'WeberC.LiuM.LiX. Q.LiuR. H.2001Antioxidant capacity of anticancer properties of red raspberry. NY Fruit Quart. 931315'},{id:"B30",body:'YangMeng.Sang-JinChung.ChinEun.Chung-OkDae.KimWon. O.SongSung. I.KooOckK.Chun2011Estimation of total antioxidant capacity from diet and supplements in US adults. Brit. J. Nut. 106254263'},{id:"B31",body:'ZhouJ. R.YuL.ZhongY.BlackburnG. L.2003Soy phytochemicals and tea bioactivity components synergistically inhibit androgen-sensitive human prostate tumors in mice. J. Nutr 133516521'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Gary M. Booth",address:null,affiliation:'
Brigham Young University, Department of Plant and Wildlife Sciences, Provo, Utah, USA
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Brigham Young University, Department of Nutrition, Dietetics, and Food Science, Provo, Utah, USA
'},{corresp:null,contributorFullName:"Christopher M. Lee",address:null,affiliation:'
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1. Introduction
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1.1 Thalassemia and hemoglobinopathies
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Thalassemia is a type of anemia caused by reduction or absence of globin chain synthesis, which results in imbalanced-globin chain synthesis; the major pathogenesis of the disease. The unaffected globin chains continuing to be synthesized at the normal synthetic rate tend to form homotetramers and aggregation that can harm the red blood cells both at young and mature stages. The α-globin chain aggregation formed in β-thalassemia causes ineffective erythropoiesis due to oxidative stress. The γ- and β-globin chain homotetramer (γ4, β4) formed in α-thalassemia harm mature erythrocytes. The γ4 or Hb Bart’s has very high oxygen affinity and inhibits oxygen release from erythrocytes which, in turn, results in tissue anoxia. The β4 or HbH is an unstable hemoglobin and precipitates easily under the stress condition in the body. Once precipitates, the erythrocytes are removed by the RE system resulting in anemia.
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Severe thalassemia cases suffer from chronic and marked anemia with life relying solely on blood transfusion. Anemia causes expansion of bone marrow, leading to osteoporosis and changes of bone structure. Blood transfusion and hemolysis cause iron overloading state in the body which causes several complications such as heart disease, growth retardation, diabetes mellitus, and infection.
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Thalassemia is considered the most common autosomal single-gene disorder worldwide. It can be found in more than 150 countries with an estimated carrier frequency of about 7%. The Mediterranean region, certain parts of North and West Africa, Middle East, Indian subcontinent, Southern Far East, and South East Asia have the highest prevalence of the disease [1].
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In contrast to the thalassemia, hemoglobinopathy is an inherited disorder of hemoglobin productions characterized by production of abnormal hemoglobin or hemoglobin structural variants occurring from genetic alterations including point mutations, deletions or insertion of the normal globin genes. The well-known abnormal hemoglobins in the world are HbS, which is common in western countries as well as in the Middle East and HbE, which is common in Southeast Asia [2].
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1.2 Types of thalassemia and hemoglobinopathies
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Two major types of thalassemia are found across the world consisting of the α- and β-thalassemia. The α-thalassemia is further sub-divided into two types: α-thalassemia 1 or αO-thalassemia and α-thalassemia 2 or α+-thalassemia. Gene deletion is the leading cause of α-thalassemia. In α-thalassemia 1, two α-globin gene in-cis on chromosome 16 are deleted, while only one α-globin gene is absent in the α-thalassemia 2 [3]. In addition, there are two types of β-thalassemia; βO-thalassemia and β+-thalassemia. In contrast to α-thalassemia, mutations on the β-globin gene are found in the majority of the β-thalassemia patients [4].
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Two broad types of hemoglobinopathies or structural variants are also found; α- and β-hemoglobinopathies. To date, approximately 1358 structural variants are described (http://globin.bx.psu.edu), around 90% of which are involved in the β, γ, and δ-globin chains and around 60% involves the α-globin chain. The most important β-globin hemoglobinopathies are HbS (α2β2\n6Glu−Val) and HbE (α2β2\n26Glu−Lys). Hb E is found around the world and accounts for approximately 13–17% on the population of Thailand, especially in the Thai-Laos-Cambodian boundary or “Hb E triangle” where more than 32–60% of the people carry HbE gene [5].
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1.2.1 β-Thalassemia
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The β-thalassemia is a diverse group of disorders of hemoglobin synthesis which is characterized by reduced or absent β-globin chain synthesis. There are two main types of β-thalassemia: βO-thalassemia in which no β-globin chain is produced and β+-thalassemia in which some β-globin is produced but less than normal.
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β0-Thalassemia is severe β-thalassemia with no production of β-globin chain. It is mainly caused by point mutations in coding region (exon) or exon-intron junction of β-globin gene which lead to premature stop codon or generation of abnormal β-globin mRNA. The end results of these abnormalities are absence of the β-globin chain production [6]. In Thailand, at least three common mutations in the β-globin gene are of this category. They comprise A-T substitution at codon 17 (CD17: A-T) which creates premature stop codon, the TTCT-deletion at codons 41/42 (CD41/42: −TTCT) which causes reading frameshift and premature stop codon at codon 59 instead of codon 147, the G-T substitution at IVSI-nt1 which leads to abnormal splicing of immature β-mRNA and results in no production of normal β-mRNA. In general, thus, genotype of heterozygote is written as β0/βA and that for homozygote as β0/β0 [2, 7, 8, 9].
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β+-Thalassemia is a milder form of β-thalassemia in which some β-globin chains are still produced. The majority of cases possess point mutations outside exons, especially in the promoter region. The mutations of β-globin gene leading to the β+-thalassemia include mutations at ATA box (nt-28, nt-29 or nt-30 from cap site), CACCC element (nt-86 to nt-90 from cap site), and mutations in introns or exons of gene to produce new splice site to race in RNA splicing process, as mutation in IVS2-nt654 (C-T) and mutation of IVS1-nt5 (G-C) [2, 6]. The genotypes were β+/βA and β+/β+ for heterozygote and homozygote consecutively.
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1.2.2 β-Hemoglobinopathies
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β-Hemoglobinopathies are characterized by the production of abnormal β-globin chains due to changes or mutations (missense mutations) on the β-globin gene. Two abnormal β-globin chains then assemble with two normal α-globin chains to form abnormal hemoglobin or β-structural variants. These abnormal hemoglobins generally have different electrophysical properties from their normal counterparts; that is, due to the molecular conformational alteration. In theory, synthetic rate of the abnormal β-globin chain should be normal. However, some are produced in reduced rate, thus producing a phenotype resembling the β+-thalassemia. To date, there are more than 737 β-globin structural variants reports across the world (http://globin.cse.psu.edu/).
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1.3 Hemoglobin E
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HbE is abnormal hemoglobin resulted from the G-A substitution at codon 26 of β-globin gene. This missense mutation partially activates a cryptic splice site toward the 3′ end of exon 1, resulting in a proportion of abnormally splice mRNA. Thus, less βE globin is synthesized, leading to a mild thalassemia phenotype. HbE is becoming the common β-globin structure variant across the world as a result of migration and inter racial marriage [2, 10]. It has been realized to be the hallmark of Southeast Asian region. In Thailand, HbE is very common accounting for approximately 8–70% of population [11].
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1.4 Inheritance of genes for β-thalassemia and HbE
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Gene for β-thalassemia and HbE is transmitted within the family from parents to descendants in an autosomal recessive fashion. Thus, those who are heterozygous for abnormal β-gene are clinically asymptomatic and called β-thalassemia carrier or β-thalassemia trait. Those who are heterozygote for HbE gene (βE) are also clinically asymptomatic and called HbE carrier or HbE trait. However, homozygote or compound heterozygote of the β-thalassemia gene and/or HbE gene are clinically affected and suffer from chronic anemia with some life-threatening complication. Therefore, accurate diagnosis of carriers of the β-thalassemia and HbE as well as the disease is important.
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1.5 Problem of β-thalassemia and HbE
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The carriers of β-thalassemia and HbE do not have clinical burden as they are clinically normal and have normal quality of life. However, if the β-thalassemia carriers get married with the HbE carriers, they will have 25% chance of producing the HbE/β-thalassemia babies. The HbE/β-thalassemia or sometimes called the β-thalassemia/HbE disease is a thalassemia syndrome that presently is known to be clinically heterogeneous [10, 11, 12]. Some patients are very mild, while some are very severe. The severe cases always required regular blood transfusion which always ends up with iron overloading condition. Without proper management of this iron overloading, several fatal complications occur, leading to low quality of life and, finally, pre-death at young age.
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1.6 Diagnosis of β-thalassemia and HbE
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The diagnosis of β-thalassemia and HbE involves both clinical and laboratory investigations. Clinical data can only identify the affected patients, but cannot 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 laboratory data can define β-thalassemia and HbE carriers.
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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 testing.
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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, 14, 15, 16]. These screening tests, however, cannot provide the information on the exact type of thalassemia of the positive persons.
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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 β-thalassemia gene. The confirmatory tests include;
Hemoglobin studies: Tests for hemoglobin studies include cellulose acetate electrophoresis, microcolumn chromatography, alkaline denaturation test, cation-exchange high performance liquid chromatography (HPLC) [17, 18, 19], cation-exchange low pressure liquid chromatography (LPLC) [20, 21, 22], capillary zone electrophoresis (CZE) [23, 24, 25, 26, 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 strip) test for Hb Bart’s [32].
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, 35, 36], and high resolution melting curve (HRM) analysis [37, 38, 39].
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2. Screening laboratory tests for β-thalassemia and HbE carriers
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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.
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2.1 Red blood cell indices
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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.
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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].
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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).
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2.2 One-tube osmotic fragility test (OFT)
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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.
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2.2.1 0.36% NaCl-based and BSS-based one-tube osmotic fragility test
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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 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.
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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).
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2.2.2 0.45% NaCl in glycerine or glycerine saline solution (GSS)-based one-tube osmotic fragility test
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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.
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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].
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2.2.3 0.45% NaCl in glycerine or glycerine saline solution (GSS)-based one-tube osmotic fragility test expressed in “hemolysis area”
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The portable spectrophotometer was invented and capable of reading absorbance and transmission of red light through the red blood cell suspension inside 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)).
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Figure 2.
Portable spectrophotometer (A) and on-screen GSS-based OFT result with the hemolysis area (HemeArea in the screen) shown in the screen (B).
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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].
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2.3 Dichlorophenolindophenol precipitation (DCIP) test
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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 βE-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].
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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).
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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].
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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).
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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.
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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].
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2.4 HbE-tube test
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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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Figure 5.
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.
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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.
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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].
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2.5 HbE screen test
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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].
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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).
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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.
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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, 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].
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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.
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3. Interpretation of the screening tests
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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.
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The MCV, MCH, and OFT are all positive in β-thalassemia diseases such as homozygous βO-thalassemia, homozygous β+-thalassemia, compound heterozygous βO/β+-thalassemia, compound heterozygous HbE/βO-thalassemia, compound heterozygous HbE/β+-thalassemia, and homozygous HbE. However, the DCIP test, HbE tube test, and HbE screen test are positive in all cases having HbE in blood.
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4. Usefulness of screening tests
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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 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.
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4.1 Confirmatory method for thalassemia and hemoglobinopathies
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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.
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4.1.1 Hemoglobin studies by cellulose acetate electrophoresis (CAE) at alkaline condition
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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.
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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.
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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 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).
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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.
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Hb patterns on CAE at pH 8.6 of adult
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HbE heterozygote (carriers)
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EA
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HbE homozygote
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EE (only 1 band of HbE)
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βO-thalassemia heterozygote (carriers)
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A2A
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β+-thalassemia heterozygote (carriers)
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A2A
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βO-thalassemia homozygote (βO/βO)
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A2F
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β+-thalassemia homozygote (β+/β+)
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A2FA
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βO/β+-thalassemia (βO/β+)
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A2FA
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HbE/βO-thalassemia (βE/βO)
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EF
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HbE/β+-thalassemia (βE/β+)
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EFA
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Table 1.
Hemoglobin pattern on CAE at pH 8.6 of β-thalassemia and HbE.
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4.1.2 Hemoglobin study by microcolumn chromatography
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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−.
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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.
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4.1.2.1 Microcolumn chromatography using DEAE-Sephadex A50 resin
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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.
Normal range of HbA2 by this protocol is 2.62 ± 0.87% [52].
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4.1.2.2 Microcolumn chromatography using DEAE-cellulose
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This protocol was described by Wood [64] and modified in the author’s laboratory in 2007 [65].
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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.
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Calculation of levels of HbA2 or HbE must follow the Eq. 2 shown below.
Normal range of HbA2 by this protocol is 1.3–3.7% (mean 2.5%) [64].
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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].
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4.1.2.3 Hemoglobin study by alkaline denaturation test of Betke
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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.
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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.
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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.
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The percentage of HbF is calculated by the following Eq. 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.
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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.
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4.1.2.4 Hemoglobin study by cation-exchange high performance liquid chromatography (HPLC)
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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).
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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 cation-exchange HPLC pattern may be also observed in carriers of α-thalassemia 1 and carriers of α-thalassemia 2.
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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%.
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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.
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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].
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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.
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4.2 Cation-exchange HPLC hemoglobin patterns of β-thalassemia carriers and HbE carriers in human adults
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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.
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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%.
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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%.
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4.3 Cation-exchange HPLC hemoglobin patterns of β-thalassemia diseases and HbE disease in human adults
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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.
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4.3.1 Homozygous βO-thalassemia
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Individuals of homozygous βO-thalassemia (βO/βO) are usually affected by the severe thalassemia disease and require regular blood transfusion. This group of patients 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).
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4.3.2 Homozygous β+-thalassemia
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Individuals of homozygous β+-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+-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+-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+-thalassemia.
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4.3.3 Compound heterozygous βO/β+-thalassemia
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Patients with compound heterozygous βO/β+-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 non-transfused cases is A2FA, being similar to homozygous β+-thalassemia (Figure 12). However, the compound heterozygous βO/β+-thalassemia has thalassemic red blood cell morphology like homozygous βO-thalassemia. In contrast, red blood cell morphology of homozygous β+-thalassemia is less abnormal than the other two β-thalassemia mentioned above.
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Figure 11.
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).
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Figure 12.
Cation-exchange HPLC hemoglobin pattern A2FA seen in homozygous β+-thalassemia and compound heterozygous β+/βO-thalassemia (HbAo: 24.6%, HbF: 68.5%, HbA2: 3.9%).
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Hb patterns by the cation-exchange HPLC of adult HbE disease also comprise several varieties depending on combination of βE mutation.
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4.3.4 Homozygous HbE
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Homozygous HbE (βE/βE) 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 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.
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4.3.5 Compound heterozygous βE and βO-thalassemia
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Individuals with compound heterozygote of βE 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).
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4.3.6 Compound heterozygous βE and β+-thalassemia
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Individuals with compound heterozygote of βE and β+-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.
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4.3.7 Double form of HbE carrier and HbH disease
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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.
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4.3.8 Double form of HbE homozygote and HbH disease
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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.
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4.3.9 Double form of HbE/βO-thalassemia and HbH disease
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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.
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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.
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Figure 14.
Hemoglobin pattern in cation-exchange HPLC of cases with HbE/βO-thalassemia (F 53.1%, E 35.8%).
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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).
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5. Hemoglobin study by capillary zone electrophoresis (CZE)
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Capillary zone electrophoresis (CZE) has been introduced for use as a tool for analysis of hemoglobin variants [23, 24, 25, 26, 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).
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Figure 16.
CZE electropherogram of normal human hemoglobins. HbA: 94.3%, HbF: 2.4%, HbA2: 3.3%.
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Figure 17.
CZE pattern of hemoglobins from zone (Z) 1 to zone (Z) 15.
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5.1 The CZE pattern of β-thalassemia carriers and HbE carriers
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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).
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Figure 18.
CZE pattern of hemoglobins of β-thalassemia carrier.
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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 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).
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Figure 19.
CZE pattern of hemoglobin of HbE carrier.
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5.2 The CZE pattern in β-thalassemia disease and HbE disease
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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 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+-thalassemia and compound heterozygous β+/βO-thalassemia, the A2FA is generally seen in CZE platform.
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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/βO-thalassemia, for example, A2EF must be reported together with the proportion of each hemoglobin (Figure 20).
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Figure 20.
CZE pattern of hemoglobins of HbE/βO-thalassemia, composing of A2EF with 4.7% HbA2, 58.9% HbE, and 36.4% HbF.
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6. Hemoglobin study by monoclonal antibody
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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].
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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.
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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]).
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7. DNA analysis for β-thalassemia and HbE
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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].
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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].
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7.1 Identification of β-thalassemia and HbE by MS-PCR
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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 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 (β17) 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.
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7.1.1 MS-PCR for β 41/42 mutation
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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 βT/βT (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/βT (A represents HbA; T represents other types of β-globin gene mutation).
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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.
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7.1.2 MS-PCR for β17 mutation
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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 β17; 5′-GGC AGA GAG AGT CAG TGC CTA-3′, 150 ng of “Normal primer” for β17; 5′-ACC TGA CTC CTG AGG AGA AGA CTG CCG TTA CTG CCC TGT GGG ACA-3′, 100 ng of “Mutant primer” for β17; 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 β17 mutation, respectively (Figure 23).
Interpretation: Samples having only 170-bp amplified fragments are homozygote for β17 with genotype β17/β17. Samples having only 190-bp fragments are negative for the β17 with genotype βA/βA or βT/βT (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 β17 with genotype of either β17/βA or β17/βT (A represents HbA; T represents other types of β-globin gene mutation).
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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.
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7.1.3 MS-PCR for βE mutation or HbE
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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 βE; 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 βE; 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 βE mutation, respectively (Figure 24).
Interpretation: Samples having only 160-bp amplified fragments are homozygote for βE with genotype βE/βE. Samples having only 138-bp fragments are negative for the βE with genotype βA/βA or βT/βT (A represents HbA; T represents other types of β-globin gene mutation). Samples having both 138 and 160-bp amplified products are heterozygote for the βE with genotype of either βE/βA or βE/βT (A represents HbA; T represents other types of β-globin gene mutation).
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Figure 24.
MS-PCR for identifying βE 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.
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8. Identification of β-thalassemia and HbE by ARMS-PCR
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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 internal control for the ARMS-PCR.
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8.1 ARMS-PCR for β41/42\n
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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.
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 5 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 439 bp is the specific amplified products, and the PCR products sizing 314 bp are the control products (Figure 25).
Interpretation: Both M and N-reactions must have the 314-bp control products and the results can be read. It both M and N-reactions have 439-bp PCR products, the samples are heterozygote for β41/42 with genotype of either β41/42/βA or β41/42/βT (A represents HbA; T represents other types of β-globin gene mutation). If the 439-bp PCR products are seen in only M-reaction, the samples are homozygote for β41/42 with genotype β41/42/β41/42. If samples have the 439-bp PCR products in only N-reaction, the samples are negative for the β41/42 with genotypes of either βA/βA or βT/βT (A represents HbA; T represents other types of β-globin gene mutation) (Figure 25).
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Figure 25.
ARMS-PCR for identifying the β41/42 mutation. The 314-bp amplified products are internal control. The 439-bp amplified products are the β41/42 specific products. Sample #1 is homozygote for β41/42 mutation as the 439-pb amplified products are seen in only “M-reaction.” Sample #3 is negative for the β41/42 mutation since the 439-bp amplified products are seen in only “N-reaction.” Sample #2 is heterozygote for the β41/42 mutation since the 439-bp amplified products are seen in both “M-reaction” and “N-reaction.”
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8.2 ARMS-PCR for β17
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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 “M17 primer”; 5′-CTC ACC ACC AAC TCA GCC ACG TTC AGC ATA-3′ (for M-reaction only), 0.15 μM of “N17 primer”; 5′-CTC ACC ACC AAC TTC ATC CAC GTT CAC ATT-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.
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 5 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 239 bp is the specific amplified products, and the PCR products sizing 314 bp are the control products (Figure 26).
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 239-bp PCR products, the samples are heterozygote for β17 with genotype of either β17/βA or β17/βT (A represents HbA; T represents other types of β-globin gene mutation). If the 239-bp PCR products are seen in only M-reaction, the samples are homozygote for β17 with genotype β17/β17. If samples have the 239-bp PCR products in only N-reaction, the samples are negative for the β17 with genotypes of either βA/βA or βT/βT (A represents HbA; T represents other types of β-globin gene mutation) (Figure 26).
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Figure 26.
ARMS-PCR for identifying the β17 mutation. The 314-bp amplified products are internal control. The 239-bp amplified products are the β17 specific products. Sample #1 is homozygote for β17 mutation as the 239-pb amplified products are seen in only “M-reaction.” Sample #3 is negative for the β17 mutation since the 239-bp amplified products are seen in only “N-reaction.” Sample #2 is heterozygote for the β17 mutation since the 239-bp amplified products are seen in both “M-reaction” and “N-reaction.”
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8.3 ARMS-PCR for βE\n
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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 “HbE-M primer”; 5′-TAA CCT TGA TAC CAA CCT GCC CAG GGC GTT-3′ (for M-reaction only), 0.15 μM of “HbE-N primer”; 5′-TAA CCT TGA TAC CAA CCT GCC CAG GGC GTC-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.
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 5 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 260 bp is the specific amplified products, and the PCR products sizing 314 bp are the control products (Figure 27).
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, the samples are heterozygote for βE with genotype of either βE/βA or βE/βT (A 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 βE with genotype βE/βE. If samples have the 260-bp PCR products in only N-reaction, the samples are negative for the βE with genotypes of either βA/βA or βT/βT (A represents HbA; T represents other types of β-globin gene mutation) (Figure 27).
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Figure 27.
ARMS-PCR for identifying βE mutation. The 314-bp amplified products are internal control. The 260-bp amplified products are the βE specific products. Sample #3 is homozygote for βE mutation as the 260-pb amplified products are seen in only “M-reaction.” Sample #1 is negative for the βE mutation since the 260-bp amplified products are seen in only “N-reaction.” Sample #2 is heterozygote for the βE mutation since the 260-bp amplified products are seen in both “M-reaction” and “N-reaction.”
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9. Identification of β-thalassemia and HbE by MAS-PCR
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This PCR technique was established by author and named multiplex allele specific (MAS)-PCR [29]\n.\n 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).
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, βE, and β17, respectively (Figure 28).
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Figure 28.
MAS-PCR for identifying β-thalassemia mutation, βE-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 βE mutation as the 293-bp amplified products are seen. Lane 6 is positive for both SEA-α thalassemia 1 and βE 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]).
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10. Pitfalls in laboratory diagnosis of β-thalassemia and HbE
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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 steps of laboratory diagnosis.
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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 results will be obtained if NaCl concentration is too high, and vice versa.\n
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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 negative results and vice versa.\n
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10.1 Pitfalls in screening tests for β-thalassemia and HbE carriers
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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.
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 screening for β-thalassemia and HbE carriers.
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.
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10.2 Pitfalls in confirmatory tests for β-thalassemia and HbE
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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 hemoglobin typing results of the patients. If the patients of β-thalassemia disease (βO/βO) have just received blood transfusion, their hemoglobin types will be A2AF, instead of A2F which is the real hemoglobin type of the βO/βO. If patients of HbE/βO-thalassemia (βE/βO) have recent blood transfusion, their hemoglobin types will be EFA, instead of EF which is the real hemoglobin type of βO/βE. Therefore, recent blood transfusion should always be taken into account when reading and interpreting the hemoglobin typing results.
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Co-existence of α-thalassemia 1 in β-thalassemia carrier and in HbE carrier can be found some regions. This is called double α-thalassemia 1/β-thalassemia carriers and double α-thalassemia 1 and HbE carriers. Results of one-tube osmotic fragility test are always positive in double α-thalassemia 1/β-thalassemia carriers, being the same as that of the single β-thalassemia carrier. However, MCV/MCH in double α-thalassemia 1/β-thalassemia carriers are slightly higher than those in the single β-thalassemia carriers (70.7 ± 2.6 vs. 68.4 ± 2.7 for MCV, 21.0 ± 1.2 vs. 20.7 ± 0.9 for MCH) [83]. HbA2 level in double α-thalassemia 1/β-thalassemia carriers is lower than that in single β-thalassemia carrier (5.27 ± 0.77% vs. 5.65 ± 0.78%) [29]. In contrast, the results of one-tube osmotic fragility test of double α-thalassemia 1/HbE carrier are usually positive, being different from those of single HbE which can be either positive or negative. The MCV/MCH in double α-thalassemia 1/HbE carrier are lower than those of the single HbE carrier (68.7 ± 8.9 fL vs. 75.7 ± 7.5 fL for MCV, 22.5 ± 3.1 pg vs. 24.9 ± 2.8 pg for MCH). HbE level in double α-thalassemia 1/HbE carrier are lower than those of the single HbE carrier (22.8 ± 4.4% vs. 27.6 ± 3.3%) [84]. Therefore, if HbE carriers have low HbE levels as well as MCV/MCH, further identification of α-thalassemia 1 genotype must be done.
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11. Conclusions
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Diagnosis of β-thalassemia and HbE requires laboratory investigations. Screening tests are used to search for carriers, while confirmatory tests are carried out in those samples positive for screening tests with the aim to make the definite diagnosis. There are several pitfalls in these laboratory tests, both in screening tests and confirmatory tests that must not be overlooked, otherwise misdiagnosis will occur.
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Acknowledgments
\n
The author thanks Mr. Eaktong Limveeraprachak of Sawan Pracharak Hospital, Nakorn Sawan Province, Thailand for providing hemoglobin typing results put in this chapter.
\n
Conflict of interest
The authors declare no conflict of interest.
\n',keywords:"β-thalassemia, HbE, screening tests, confirmatory tests, thalassemia carrier, HbE carrier, β-thalassemia disease",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/70780.pdf",chapterXML:"https://mts.intechopen.com/source/xml/70780.xml",downloadPdfUrl:"/chapter/pdf-download/70780",previewPdfUrl:"/chapter/pdf-preview/70780",totalDownloads:655,totalViews:0,totalCrossrefCites:0,dateSubmitted:"May 14th 2019",dateReviewed:"October 30th 2019",datePrePublished:"January 10th 2020",datePublished:"September 23rd 2020",dateFinished:"January 10th 2020",readingETA:"0",abstract:"β-Thalassemia and HbE, each, is a syndrome resulted from quantitative and qualitative defects of β-globin chain, respectively. In addition to history retrieve and physical examination, diagnosis of these disorders requires laboratory information. Laboratory tests that are conventionally performed to diagnose the β-thalassemia and HbE are classified into two groups, based on the purposes, including the screening tests and confirmatory tests. The screening tests are aimed to screen for carriers of the β-thalassemia and HbE, while confirmatory tests are the tests performed to definitely diagnose these disorders. This chapter will explain all of these tests, the information of which will be useful for those who are working and interested in the β-thalassemia and HbE.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/70780",risUrl:"/chapter/ris/70780",signatures:"Thanusak Tatu",book:{id:"8450",title:"Beta Thalassemia",subtitle:null,fullTitle:"Beta Thalassemia",slug:"beta-thalassemia",publishedDate:"September 23rd 2020",bookSignature:"Marwa Zakaria and Tamer Hassan",coverURL:"https://cdn.intechopen.com/books/images_new/8450.jpg",licenceType:"CC BY 3.0",editedByType:"Edited by",isbn:"978-1-83880-587-6",printIsbn:"978-1-83880-586-9",pdfIsbn:"978-1-83880-588-3",editors:[{id:"187545",title:"Prof.",name:"Marwa",middleName:null,surname:"Zakaria",slug:"marwa-zakaria",fullName:"Marwa Zakaria"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_1_2",title:"1.1 Thalassemia and hemoglobinopathies",level:"2"},{id:"sec_2_2",title:"1.2 Types of thalassemia and hemoglobinopathies",level:"2"},{id:"sec_2_3",title:"1.2.1 β-Thalassemia",level:"3"},{id:"sec_3_3",title:"1.2.2 β-Hemoglobinopathies",level:"3"},{id:"sec_5_2",title:"1.3 Hemoglobin E",level:"2"},{id:"sec_6_2",title:"1.4 Inheritance of genes for β-thalassemia and HbE",level:"2"},{id:"sec_7_2",title:"1.5 Problem of β-thalassemia and HbE",level:"2"},{id:"sec_8_2",title:"1.6 Diagnosis of β-thalassemia and HbE",level:"2"},{id:"sec_10",title:"2. Screening laboratory tests for β-thalassemia and HbE carriers",level:"1"},{id:"sec_10_2",title:"2.1 Red blood cell indices",level:"2"},{id:"sec_11_2",title:"2.2 One-tube osmotic fragility test (OFT)",level:"2"},{id:"sec_11_3",title:"2.2.1 0.36% NaCl-based and BSS-based one-tube osmotic fragility test",level:"3"},{id:"sec_12_3",title:"2.2.2 0.45% NaCl in glycerine or glycerine saline solution (GSS)-based one-tube osmotic fragility test",level:"3"},{id:"sec_13_3",title:"2.2.3 0.45% NaCl in glycerine or glycerine saline solution (GSS)-based one-tube osmotic fragility test expressed in “hemolysis area”",level:"3"},{id:"sec_15_2",title:"2.3 Dichlorophenolindophenol precipitation (DCIP) test",level:"2"},{id:"sec_16_2",title:"2.4 HbE-tube test",level:"2"},{id:"sec_17_2",title:"2.5 HbE screen test",level:"2"},{id:"sec_19",title:"3. Interpretation of the screening tests",level:"1"},{id:"sec_20",title:"4. Usefulness of screening tests",level:"1"},{id:"sec_20_2",title:"4.1 Confirmatory method for thalassemia and hemoglobinopathies",level:"2"},{id:"sec_20_3",title:"Table 1.",level:"3"},{id:"sec_21_3",title:"4.1.2 Hemoglobin study by microcolumn chromatography",level:"3"},{id:"sec_21_4",title:"4.1.2.1 Microcolumn chromatography using DEAE-Sephadex A50 resin",level:"4"},{id:"sec_22_4",title:"4.1.2.2 Microcolumn chromatography using DEAE-cellulose",level:"4"},{id:"sec_23_4",title:"4.1.2.3 Hemoglobin study by alkaline denaturation test of Betke",level:"4"},{id:"sec_24_4",title:"4.1.2.4 Hemoglobin study by cation-exchange high performance liquid chromatography (HPLC)",level:"4"},{id:"sec_27_2",title:"4.2 Cation-exchange HPLC hemoglobin patterns of β-thalassemia carriers and HbE carriers in human adults",level:"2"},{id:"sec_28_2",title:"4.3 Cation-exchange HPLC hemoglobin patterns of β-thalassemia diseases and HbE disease in human adults",level:"2"},{id:"sec_28_3",title:"4.3.1 Homozygous βO-thalassemia",level:"3"},{id:"sec_29_3",title:"4.3.2 Homozygous β+-thalassemia",level:"3"},{id:"sec_30_3",title:"4.3.3 Compound heterozygous βO/β+-thalassemia",level:"3"},{id:"sec_31_3",title:"4.3.4 Homozygous HbE",level:"3"},{id:"sec_32_3",title:"4.3.5 Compound heterozygous βE and βO-thalassemia",level:"3"},{id:"sec_33_3",title:"4.3.6 Compound heterozygous βE and β+-thalassemia",level:"3"},{id:"sec_34_3",title:"4.3.7 Double form of HbE carrier and HbH disease",level:"3"},{id:"sec_35_3",title:"4.3.8 Double form of HbE homozygote and HbH disease",level:"3"},{id:"sec_36_3",title:"4.3.9 Double form of HbE/βO-thalassemia and HbH disease",level:"3"},{id:"sec_39",title:"5. Hemoglobin study by capillary zone electrophoresis (CZE)",level:"1"},{id:"sec_39_2",title:"5.1 The CZE pattern of β-thalassemia carriers and HbE carriers",level:"2"},{id:"sec_40_2",title:"5.2 The CZE pattern in β-thalassemia disease and HbE disease",level:"2"},{id:"sec_42",title:"6. Hemoglobin study by monoclonal antibody",level:"1"},{id:"sec_43",title:"7. DNA analysis for β-thalassemia and HbE",level:"1"},{id:"sec_43_2",title:"7.1 Identification of β-thalassemia and HbE by MS-PCR",level:"2"},{id:"sec_43_3",title:"7.1.1 MS-PCR for β 41/42 mutation",level:"3"},{id:"sec_44_3",title:"7.1.2 MS-PCR for β17 mutation",level:"3"},{id:"sec_45_3",title:"7.1.3 MS-PCR for βE mutation or HbE",level:"3"},{id:"sec_48",title:"8. Identification of β-thalassemia and HbE by ARMS-PCR",level:"1"},{id:"sec_48_2",title:"8.1 ARMS-PCR for β41/42\n",level:"2"},{id:"sec_49_2",title:"8.2 ARMS-PCR for β17",level:"2"},{id:"sec_50_2",title:"8.3 ARMS-PCR for βE\n",level:"2"},{id:"sec_52",title:"9. Identification of β-thalassemia and HbE by MAS-PCR",level:"1"},{id:"sec_53",title:"10. Pitfalls in laboratory diagnosis of β-thalassemia and HbE",level:"1"},{id:"sec_53_2",title:"10.1 Pitfalls in screening tests for β-thalassemia and HbE carriers",level:"2"},{id:"sec_54_2",title:"10.2 Pitfalls in confirmatory tests for β-thalassemia and HbE",level:"2"},{id:"sec_56",title:"11. Conclusions",level:"1"},{id:"sec_57",title:"Acknowledgments",level:"1"},{id:"sec_60",title:"Conflict of interest",level:"1"}],chapterReferences:[{id:"B1",body:'\nWeatherall DJ, Clegg JB. The Thalassaemia Syndromes. 4th ed. Oxford: Blackwell Scientific; 2001\n'},{id:"B2",body:'\nFlint J, Harding RM, Boyce AJ, Clegg JB. The population genetics of the haemoglobinopathies. In: Rodgers GP, editor. Bailliere’s Clinical Haematology, International Practice and Research: Sickle Cell Disease and Thalassaemia. Vol. 11. London: Bailliere Tindall; 1998. pp. 1-51\n'},{id:"B3",body:'\nHiggs DR, Weatherall DJ. 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Annals of the Academy of Medicine, Singapore. 1996;25(1):75-78\n'},{id:"B73",body:'\nJintaridth P, Srisomsap C, Vichittumaros K, Kalpravidh RW, Winichagoon P, Fucharoen S, et al. Chicken egg yolk antibodies specific for the gamma chain of human hemoglobin for diagnosis of thalassemia. International Journal of Hematology. 2006;83(5):408-414\n'},{id:"B74",body:'\nShyamala M, Kiefer CR, Moscoso H, Garver FA. A monoclonal antibody-linked immunoassay for hemoglobin H disease. Annals of Hematology. 1992;65(1):37-40\n'},{id:"B75",body:'\nWinichagoon P, Kumpan P, Holmes P, Finlayson J, Newbound C, Kabral A, et al. Validation of the immunochromatographic strip for alpha-thalassemia screening: A multicenter study. Translational Research. 2015;165(6):689-695\n'},{id:"B76",body:'\nShyamala M, Kiefer CR, Moscoso H, Garver FA. Application of a monoclonal antibody specific for the delta chain of hemoglobin A2 in the diagnosis of β thalassemia. American Journal of Hematology. 1991;38(3):214-219\n'},{id:"B77",body:'\nNeeds T, Lynch DT. Beta Thalassemia. Treasure Island, FL: StatPearls; 2019\n'},{id:"B78",body:'\nThein SL. Genetic basis and genetic modifiers of beta-thalassemia and sickle cell disease. Advances in Experimental Medicine and Biology. 2017;1013:27-57\n'},{id:"B79",body:'\nOld J, Varawalla NY, Weatherall DJ. Rapid detection and prenatal diagnosis of β-thalassemia: Studies in Indian and Cypriot population in the UK. Lancet. 1990;336:834-837\n'},{id:"B80",body:'\nSritong W, Tatu T, Saetung R, Sanguansermsri T. Rapid identification of β-thalassemia mutations by mutagenically separated polymerase chain reaction. Chiangmai Medical Bulletin. 2004;43(4):133-141\n'},{id:"B81",body:'\nSirichotiyakul S, Saetung R, Sanguansermsri T. Analysis of β-thalassemia mutations in northern Thailand using an automated fluorescence DNA sequencing technique. Hemoglobin. 2003;27(2):89-95\n'},{id:"B82",body:'\nSuwannasin S. Study of Molecular Background of Hematologically Silent α-Thalassemia and Development of Polymerase Chain Reaction to Detect Double Heterozygous α/β Thalassemia Commonly Found in Thais. Chiang Mai University: Chiang Mai University; 2009\n'},{id:"B83",body:'\nTatu T, Sayachak S, Chiampanichayakul S, Kasinrerk W, Piyamongkol W. Red blood cell parameters and Hb Bart’s levels in simple β-and double α/β-thalassemia heterozygotes. In: Thai Society of Hematology Annual Meeting March 26-29, 2006; The Royal Golden Jubilee Building, Bangkok. 2006\n'},{id:"B84",body:'\nLeckngam P, Limweeraprajak E, Kiewkarnkha T, Tatu T. The Hb E (HBB: c.79G>A), mean corpuscular volume, mean corpuscular hemoglobin cutoff points in double heterozygous Hb E/− −(SEA) alpha-Thalassemia-1 carriers are dependent on hemoglobin levels. Hemoglobin. 2017;41(1):38-43\n'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Thanusak Tatu",address:"tthanu@hotmail.com;, thanusak.t@cmu.ac.th",affiliation:'
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