Safe Blood Transfusion in Beta-Thalassemia

#### **Chapter 5**

## Novel Diagnostic Approach and Safe Blood Transfusion Practices for Thalassemia: A Vital Role of a Blood Centre in Western India

*Avani Shah, Sumit Bharadva, Parizad Patel and Kanchan Mishra*

### **Abstract**

β-Thalassemia carrier's frequency is uneven in different districts in Gujarat (0–9.5%). Surat Raktadan Kendra & Research Centre (SRKRC), NABH accredited and regional blood Centre in Surat (Gujarat, India) running since 1976, provides free blood units to more than 350 Thalassemia Major Patients every year. Our DSIR (Department of Scientific and Industrial Research, Ministry of Science and Technology, Govt. of India) approved Research department has developed Multiplex ARMS-PCR including 4 common mutations which is a fast, reliable, and cost-effective method. Safe blood transfusion is a priority for these patients because of having transfusion-associated infections, formation of alloantibodies against donor's antigens, developing different grades of Blood Transfusion Reactions (BTR's), etc. Therefore, different approaches have been implemented as routine practice by our Blood Centre, like the use of saline washed and/or leuco-reduced Red Cell Concentrate for transfusion (reduces the risk of BTR's), NAT testing for Transfusion Transmitted Infections (reduces window period of TTI's), antibody screening of patient (if develop in patient) and molecular genotyping of clinically important blood group antigens (Difficult to type these patients serologically due to presence of donors' red cells). Despite being Blood Centre, contributing to developing novel diagnostic techniques and strictly following all possible transfusion practices, SRKRC greatly helps in ensuring proper diagnosis, lengthening the transfusion period and providing the safest blood to these patients.

**Keywords:** β-Thalassemia in India, multiplex ARMS-PCR, NAT testing, molecular genotyping, blood transfusion, alloantibodies

#### **1. Introduction**

From a historical perspective, it is always a question and debate in what way β-thalassemia has spread in the world to reach such high incidences in certain populations. Hemoglobinopathies, including hemoglobin variants and thalassemia, are a group of inherited disorders that arise due to mutation and/or deletions of one or more globin genes that result in the production of structurally abnormal Hb variants in the former and reduced rate of synthesis of the normal globin chains in the latter [1, 2]. Thalassemia is a group of disorders of hemoglobin in which either

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globin chain synthesis is reduced or absent, therefore it is termed as a quantitative disorder [3]. This inherited disorder of hemoglobin is considered the commonest single-gene disorder globally with an autosomal recessive inheritance, which estimated around 300,000 to 400,000 babies born each year suffering from the same [4]. It is increasingly prevalent in the Mediterranean, Asia and Sub-Saharan Africa along with other continents such as Europe, North America, and Australia due to population migration and therefore, has become a global health problem [5]. The carrier frequencies for β-thalassemia in these areas range from 1 to 20%, and rarely may be higher. The frequencies for the milder forms of β-thalassemia are much greater, varying from 10 to 20% in parts of sub- Saharan Africa to 40% or more in some Middle Eastern and Indian populations, to as high as 80% in northern Papua New Guinea and some isolated groups in the northeast of India [4].

#### **1.1 The hemoglobinopathies: Burden in India**

Among the different hemoglobinopathies, Beta-thalassemias and Sickle Cell Disorders (SCD) pose a significant health burden in India. The average prevalence of β-thalassemia carriers is 3–4%, which translates to 35 to 45 million carriers in our multi-ethnic and culturally and linguistically diverse population of 1.21 billion people that also includes around 8% of tribal groups. Several ethnic groups have a much higher prevalence (4–17%) [6, 7]. Every year 10,000 children are being born with thalassemia which approximately accounts for 10% of the total world incidence of thalassemia-affected children [8] and one in eight thalassemia carriers live in India. There are nearly 42 million carriers of the β-thalassemia trait. There are communities in which it is more prevalent like Sindhis, Punjabis, Gujaratis, Bengalis, Mahars, Kolis, Saraswats, Lohanas, and Gaurs [9]. However, in the absence of National Registries of patients, the exact numbers are not known. The March of Dimes Global Report on Birth Defects has estimated that the prevalence of pathological hemoglobinopathies in India is 1.2 per 1000 live births. It has been suggested that there would be 32,400 babies with a serious hemoglobin disorder born each year based on 27 million births per year in India [10–12]. Of the 10,000 to 12,000 thalassemic children born annually in India, very few are optimally managed mainly in urban regions although the Government of India has included the care and management of patients with thalassemia and sickle cell disease in the 12th Five Year Plan. It has been estimated that 2 million units of packed red cells would be needed for transfusion of thalassemia patients in the country [13, 14]. Better management for beta-thalassemia major patients mainly in urban regions in India with regular and safe blood transfusions and adequate iron chelation allows them to have a better quality of life.

Hemoglobinopathies are more common in Gujarat compared to other Indian states. Model and Petrou [15] have estimated a 12% incidence of major hemoglobinopathy traits in Gujarat. Several studies have revealed a high prevalence of β-thalassemia trait (BTT) in some caste groups in Gujarat [16–18]. Certain castes and tribes in Gujarat are yet to be investigated for thalassemia and other abnormal haemoglobins. About 10–15% of the tribal population of India is in Gujarat, particularly in South Gujarat and the prevalence of sickle cell trait (SCT) varies from 0 to 31.4% among different tribes [19, 20].

#### **2. Counseling, screening and prevention of beta-thalassemia**

Diagnosis and management of this disorder both in adults and in newborns, using appropriate approaches and uniform technology are important in different regions of a vast and diverse country like India. SRKRC is running a screening and *Novel Diagnostic Approach and Safe Blood Transfusion Practices for Thalassemia… DOI: http://dx.doi.org/10.5772/intechopen.101672*

prevention program for Beta-thalassemia and Sickle cell anemia for the last 15 years. Under this program, students from colleges and universities are screened for hemoglobinopathies. The prevalence of Beta-thalassemia trait (BTT) was 4.4% and sickle cell anemia was 1.3%, reported by SRKRC [20]. Castes like Lohana (10.8%), Sindhi (10.2%), Prajapati (6.3%) and Ghanchi (6.2%) had a higher prevalence of BTT, whereas Gamit, Vasava, Chaudhary and Mahyavanshi had a higher prevalence of BTT (15.9, 13.6, and 6.9%) and SCT (22.2, 15.2, and 4.2%) respectively [20]. This prevalence rate is not much different from the other parts of the country. Surat is a multiethnic city. Castes like Lohana, Sindhi, Prajapati, Ghanchi, Gamit, Vasava, Chaudhary, and Mahyavanshi have a high prevalence of thalassemia and sickle cell anemia are prevalent. In Surat city, the prevalence of Beta- thalassemia trait was 4.4% and sickle cell anemia was 1.3%, reported by SRKRC.

#### **2.1 Counseling for hemoglobinopathies**

In every part of the world, there are individual groups that require counseling for screening and diagnosis of their disease status. Such groups are [21];


#### *2.1.1 Pre-marriage counseling and screening*

Undiagnosed Hemoglobinopathies is a potential threat, pre-marriage counseling and screening is quick way that brings immediate change and result. In India still, 50% of the marriages take place where a girl and a boy do not know each other. In such premarital counseling can be difficult. Premarital counseling can be done after screening the carrier state of an individual and making him/her understand the importance of this test and future consequences. Counseling can be done by preparing booklets, short movies and posters to increase awareness in society. The marriage register can also do premarital consoling when any marriage gets registered at the marriage registrar's office. In villages, the main leaders or panchayatmukhiya can take the responsibility of counseling and increase awareness among the people. Furthermore, some studies found the increased rate of marriage cancelation due to a better understanding of the disease and counseling by the couples at risk [22]. However, the actual targets have not been achieved yet due to many reasons including consanguineous marriages, screening just before the marriage, individual or family commitment, non-availability of alternative suitable partner, etc.

#### *2.1.2 Antenatal screening*

It is very important in cases where premarital testing has not been done. Antenatal screening usually takes place roughly between 12- and 20-weeks gestations of

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pregnancy. This screening is a sandwich between premarital screenings, counseling and prenatal diagnosis. After the antenatal screening, if both the partners found minor for Beta- thalassemia or sickle cell anemia, proper counseling should be done for prenatal diagnosis. When a thalassemia major child takes birth, to give him/her quite healthy and stable life a family needs to bear almost 2 lakhs per year including blood transfusion and medications. Even after spending such a huge amount of money and other pain full procedures of blood transfusion, a major child may not live more than 20–30 yrs. We can prevent the birth of such a child with the help of prenatal diagnosis by spending 10,000 rupees. By preventing the birth of a thalassemia major child, we can get rid of pain and disappointment.

#### *2.1.3 Preconception counseling and screening*

The infertility rate has increased due to stress, unhealthy lifestyles, marriages at older age, etc. that ultimately enhances procedures like Intra-Uterine Insemination (IUI) and In-vitro Fertilization (IVF). Both these procedures should be done under the medical council guidelines. However, in India, it is very difficult where even pregnant women do not register in clinics before 12 weeks of gestation. In the case of preconception, counseling and screening are very important as if anyone partner is minor; another partner of egg/sperm donor must be screened for the same before conception.

#### *2.1.4 Prenatal diagnosis*

The purpose of prenatal hemoglobinopathy screening is to identify and counsel asymptomatic individuals whose offspring are at risk of an inherited hemoglobinopathy. Although, prenatal diagnosis is a huge set-up and sustains process that needs expertise in molecular biology techniques. Proper and effective counseling is the key factor for prenatal diagnosis. A couple who are not screened for Betathalassemia minor and diagnosed at the time of pregnancy, for such kind of couple would be requiring prenatal diagnosis within 3 months of pregnancy. After the result of prenatal diagnosis, if the unborn is diagnosed as thalassemia major, a gynecologist may give an option of abortion to such a couple.

#### *2.1.5 Neonatal screening*

Universal screening should be done where all newborn babies in these highrisk groups are screened, as this would allow identification of other clinically significant disorders such as homozygous Beta-thalassemia and all cases of Hb S–Beta-thalassemia that could be transfusion dependent. Minor couples who do not go for prenatal diagnosis, their newborn should be tested for mutation of thalassemia major.

#### *2.1.6 Genetic counseling*

Once a thalassemia major baby is born in a family it is very important to make the parents and the family understands the consensus of the disease. Genetic guidance is an effective preventive and educational process that improved the quality of life of patients, preventing complications and sequels and allowed the referral of those who may transmit altered genes for clinical diagnosis and to genetic counseling services. A trained counselor, a hematologist or a pediatrician can effectively do this job. It is always a challenge for a family to raise such a diseased child like a normal one, moreover to bear the mental, physical and economical trauma lifelong that will be associated with the blood transfusions and other treatments for the child.

#### **2.2 Methodologies for Hemoglobinopathy screening and diagnosis: guidelines for laboratories**

For screening and detection of hemoglobinopathies especially the minor cases, certain guidelines have been prepared nationally and internationally [21, 23–28]. In India, the Ministry of Health and Welfare, Government of India issued "Guidelines on hemoglobinopathies in India" in the year 2016 [28]. Before screening test, individuals' family history, clinical evaluation if any, family origin, basic details like age, birth date, gender, the occupation should be recorded in the form of informed consent. A basic test like complete blood count (CBC) on an automated or semi-automated analyzer along with the stained peripheral blood smear should be assessed primarily. Abnormal red cell morphologies like microcytosis, iron deficiency anemia and anemia of chronic disease should be ruled out and investigations would be carried out for thalassemia with considering other hemoglobinopathies as well.

An automated cation exchange High-Performance Liquid Chromatography (HPLC) is the method of choice for hemoglobin estimation. HPLC gives an accurate estimation and quantification of different haemoglobins (Hb) like Hb A2, Hb F, Hb A and also the detection of Hb variants like HbS, HbD, HbE, HbD Punjab, etc. Despite the high cost of HPLC machines, many centres in India have this facility. The accuracy of HPLC is very high than that of the electrophoresis processes which is a more manual method compared to HPLC. Methods like isoelectric focusing, capillary and paper electrophoresis were earlier choices to use, now not used extensively in India. Followings are the screening tests used for thalassemia and other hemoglobinopathies.

#### *2.2.1 Naked eye single tube red cell osmotic fragility test (NESTROFT)*

This test is based on the osmotic fragility of red blood cells (RBCs) using the 0.36% buffered saline and has been used as a preliminary test particularly for the Beta-thalassemia carriers in India. The excessive use of this test in India may be because of the low cost, no need for any specialized equipment, and result within 10 minutes though the test gives false results due to very low specificity, false positivity, and false negativity in many clinical conditions like iron deficiency anemia where RBCs become fragile. Especially it is not recommended where automated cell counters are available. However, the primary investigation of large population in the rural area with fewer facilities would be carried out with this test.

#### *2.2.2 Red blood cell indices- complete blood count (CBC)*

There are substantial changes are happening in the RBC indices in the case of Beta-thalassemia minor with Mean corpuscular volume (MCV) <80 fl and Mean corpuscular hemoglobin concentration (MCH) <27 pg. with usually high RBC counts for the level of hemoglobin. Deviations in indices occur when samples are not run within a few hours. In such a case MCH is a more stable and reliable parameter than MCV. It is not always the case that each Beta-thalassemia minor has low MCV, MCH and high RBC count, Atypical Beta-thalassemia minor may show completely normal indices which can be missed during screening.

#### *2.2.3 Quantization of HbA2*

Hb A2 levels >4.0% along with low MCV, MCH and higher RBC counts are considered as the classical case of Beta-thalassemia carrier. HPLC being the method of choice gives an accurate quantification of Hb A2. Correct interpretations of

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borderline HbA2 levels (3.3–3.9%) are of utmost importance and need to be done with great caution. Such borderline cases should be confirmed by the DNA-based diagnostic methods. Mutations in the promoter region or the poly-A tail of the b-globin gene may have the completely normal RBC indices and/or HbA2 in the Beta-thalassemia carriers. **Figures 1** and **2** depict the retention time graph of different hemoglobins on HPLC (Figures obtained from our Research department).

#### **Figure 1.**

*HPLC report of with normal levels of Hb A2.*

**Figure 2.** *HPLC report of high levels of Hb A2: A case of Beta-thalassemia.*

*Novel Diagnostic Approach and Safe Blood Transfusion Practices for Thalassemia… DOI: http://dx.doi.org/10.5772/intechopen.101672*

Quantification of other hemoglobin variants apart from Hb A2 is also important to know the heterozygosity with two Hb variants like Beta-thalassemia carriers + sickle cell anemia carriers, Beta-thalassemia carriers + Hb E carriers, Betathalassemia carriers + Hb D carriers, etc. HPLC results with >10%, HbA2% is suggestive of the presence of other Hb variants like HPLC also quantifies Hb S, Hb D Punjab and Hb E and some fewer common variants which may elute in the P3 window (Hb J variants) or as unknown peaks.

Many large studies, either hospital-based or population screening programs for identification of hemoglobinopathies using HPLC analysis have been reported from India recently [12–14, 25, 29–32]. A multicenter study on screening university students and pregnant women in six states (Maharashtra, Gujarat, Karnataka, West Bengal, Assam, and Punjab) showed that the prevalence of Beta-thalassemia varied from 1.48% to 3.64% [12].

A large study on screening for hemoglobinopathies among non-tribal and tribal populations from different cities in Gujarat showed an overall prevalence of Beta-thalassemia trait of 1.95% and HbS trait of 6.5%. A high prevalence of Beta-thalassemia trait was seen among the Bhanushalis (8.1%), Bhakta (7.9%) and Lohanas (6.5%) [20]. A study of 65,779 cases by HPLC from Maharashtra reported 11.2% of BTT and 2.2% of SCT [33]. Tribal groups in Maharashtra have shown a prevalence of the Beta-thalassemia trait of 1.6 to 5.6% [34] while the prevalence of Beta-thalassemia trait has also been high (6.3 to 8.5%) among some tribal groups in Orissa [35] and in the non-tribal populations of Madhya Pradesh in Central India (9.59%) [36]. Thus, Beta-thalassemia is not uncommon among many non-tribal and tribal populations in India.

#### *2.2.4 Cellulose acetate electrophoresis*

This is completely a manual technique starting from preparing the hemolysate to the quantifying bands that appear on the cellulose acetate membrane at alkaline pH. Such manual technique required adequate experience and high skills. The technique is not used at those centres where HPLC is available as it's a manual, time-consuming and cumbersome method and results may vary from person to person though it is highly cost-effective compared to HPLC.

#### *2.2.5 Quantization of Hb F*

Quantification of Hb F (Fetal hemoglobin) is extremely important to identify diseased and carrier conditions. HPLC is one of the most widely used methods for this purpose. Earlier alkali denaturation was extensively used for the same. Hb F is the important parameter for the Indian population to differentiate between Betathalassemia and iron deficiency anemia because later is also more prevalent in India. When Hb F is being enumerated using HPLC, it needs to be ensured that the peak coming into the Hb F window is Hb F and not another Hb variant. **Figure 3** shows the diagrammatic view of screening carrier cases of hemoglobinopathies.

#### *2.2.6 Molecular methods for the confirmation of mutations*

Common Polymerase Chain Reaction (PCR) based detection techniques used for identifying the Beta-thalassemia point mutations are allele-specific PCR, reverse dot blot (RDB) analysis, real-time PCR with melting curve analysis, and DNA sequencing [28, 37]. DBS (Dried blood spot) filter paper matrix is required for the extraction of DNA from the whole blood sample which includes crude boiling preparation, alkali denaturation and other kit-based methods. As per the laboratory

**Figure 3.** *Screening of Beta-thalassemia carriers and other haemoglobinopathies [37].*

need and setup, the best methods of DNA extraction for PCR-based analysis may be selected [38]. PCR-based molecular assays are extremely susceptible to the aerosol contamination of amplicons. To minimize this one way-directional workflow is required that is amplification and analysis must be done in the separated rooms/ labs with all the necessary and safety precautions [39]. A positive control (having genotype positive DBS or known samples for heterozygous or homozygous hemoglobinopathies), negative control and a regent control (no template control) must be run with each PCR protocol.

#### *2.2.6.1 Allele-specific PCR*

This technique employs two primers identical in sequence except for the 30- terminus base, one of which is complementary to the wild type and the other for the mutant base; a common primer for the opposite strand must of course be used as well [37]. For primer extension to occur using Taq polymerase which has no 30–50 exonuclease (proofreading) activity, perfect matching of the primer 30-terminus with the DNA template must occur. With a normal individual, the PCR product will be seen only in the reaction employing the wild-type primer set. A heterozygote will generate a band using both wild type and mutant primer set, and an individual with a homozygous mutation will be negative with the normal and positive with the mutant primer set.

#### *2.2.6.2 Reverse dot-blot hybridization*

This is quite a routine procedure identifying suspected mutation using hybridization of an allele-specific oligomer (ASO) DNA probe [37]. For each mutation, two hybridization reactions are conducted, one with the probe for the mutant sequence and the normal sequence. The stringency of hybridization has to be optimized for each ASO probe. The ASO probes have an amino group at the 50-terminal base that enables them to attach to the nylon membrane strip after this hybridization takes place with amplified DNA that is labeled with biotin. Normal allele gives develop dots with each wild type probe but not with any mutant probe.

*Novel Diagnostic Approach and Safe Blood Transfusion Practices for Thalassemia… DOI: http://dx.doi.org/10.5772/intechopen.101672*

**Figure 4.**

*Detection of common Indian Beta-globin gene mutation by CRDB (source: Obtained from in-house experiment).*

Minor/carrier/heterozygotes give one mutation dot and one normal dot, whereas major/homozygotes give dot with the only mutated probe. Being a semi-automated method critical care should be taken at the time of washing the blots and optimization is required that can be achieved by the optimizing ASO probe length. **Figure 4** shows the in-house developed blots of different thalassemia mutations.

#### *2.2.6.3 Novel approach: multiplex ARMS-PCR for detecting 4 common Beta-thalassemia mutations*

The analysis of transfusion-dependent thalassemia major cases, attending our centre, suggested that there is a high prevalence in Muslims, Patels, Sindhis, ModhBanias, and Mahayavanshi [40–42]. Certain sub-castes of Patel [19] and tribal communities are already studied for sickle cell disorders [43, 44]. Dhodia Patel, the third largest tribal group in Gujarat, needs b-thalassemia studies as thalassemia major cases are identified in this community [41, 42].

The purpose of the present investigation was to establish and standardize a multiplex-ARMS procedure to detect ethnic-specific common mutations like IVS I-5 (G/C), Codon 41/42 (-TCTT), 619-bp deletion and FS 8/9 (þG) in one tube. This method is very convenient to screen the most commonly known molecular defects in a single Multiplex ARMS-PCR tube and detection on agarose gel electrophoresis based on specific PCR product size for each mutation [45–48].

Our in-house developed method was subsequently tested on 110 unrelated samples with unidentified Beta-thalassemia mutations. The codons IVS 1–5 mutation was the most common beta-thalassemia mutation in the Surat population. The following mutations were presented in decreasing frequency: IVS 1e5 (G/C) < Codon 8/9 (þG) < Codon 41/42 (-CTTT) < 619 bp deletion. **Figure 5** shows the amplification of 4 common mutations on an agarose gel stained with ethidium bromide. This ARMS multiplex system was found reliable, cost-effective, fast and most applicable for mutation screening of Thalassemia in Surat populations [40].

#### *2.2.6.4 A comprehensive next-generation sequencing (NGS) platform for screening and genotyping in subjects with Hemoglobinopathies*

The next advance in molecular diagnostics for hemoglobin disorders will be next-generation sequencing. Recently, NGS has been introduced to screen for thalassemia. More loci including genetic modifiers which have significant effects on clinical manifestation should be covered in the NGS screening, which is important for precise diagnosis and treatment of thalassemia [49–51].

**Figure 5.** *3% agarose gel showing the multiplex ARMS-PCR for four common mutation of Beta-thalassemia.*

There will be some technical challenges in implementing next-generation sequencing, especially for the HBA genes, which, because of the nearly identical sequence between the HBA1 and HBA2 genes, will make it challenging to determine whether a given mutation belongs to one or the other of these genes. We can hope that the cost of these technologies will eventually decrease enough to make them available to resource-limited settings where the diagnosis of hemoglobin disorders will be most valuable.

#### *2.2.6.5 Other DNA based methods*

Several other methods are used apart from these above-mentioned. In that Realtime PCR with melting curve analysis, Direct DNA sequencing, multiplex ligationdependent probe amplification (MLPA) and Next-generation sequencing have been used. RT-PCR or quantitative PCR eliminates the post PCR steps, time consumption and labour intensiveness of conventional PCRs [52, 53]. If any novel or rare mutation is present in the population, direct DNA sequencing would be the best method for the same that uses streptavidin-coated magnetic beads [54, 55]. MLPA allows the detection of any deletions or duplications in the screened regions. It requires only thermocycler and CE equipment [30, 56]. Limitations of conventional thalassemia diagnosis methods are missed diagnoses due to normal or borderline red blood cell indices and/or Hb A2 levels, various labour-intensive methods may need to identify disease-causing mutation for thalassemia that have more than 1800 mutations ranging from point mutation to large deletion.

#### **2.3 Prevention and control of Beta-thalassemia**

#### *2.3.1 Creating awareness in the general population*

The success of the prevention and control of any disease depends upon the awareness among the general population and how well the population is educated about the same [57]. In India, many social welfare clubs like Rotary, Lions, various NGOs, even Thalassemia Parents-Patients societies have been conducting education and awareness programs. Yet, awareness about b thalassemia among pregnant women in 6 states in the multi-centric Jai Vigyan programme was very limited varying from 0.2% to 4.8% in Bangalore, Vadodara, Mumbai, Dibrugarh and Ludhiana and 20.7% in Kolkata [25]. More than 50% of the rural population has not heard about thalassemia or they have some misconceptions and myths regarding this [34], whereas the majority of the people of urban areas were not ready for pre-marital screening [35]. Yet thalassemia education has not been included in the high school

curriculum in India. However, this strategy has worked in the Mediterranean region [6]. The use of mass media would create a great impact over a longer period by repeatedly showing the short films or programs on thalassemia [36].

#### *2.3.2 Target population screening for prevention and control*

For every country and population single strategy may not work all the time [28]. Defining and selecting the target population and their screening on time are the important aspects for possible prevention and control. **Table 1** shows the different timings and the age groups where screening can be possible.

### **3. Role of a blood center in the management of β-thalassemia patients**

A Blood center plays a crucial role in providing safe blood particularly to those patients for whom RBC transfusion is the principal support to live. As stated by the World Health Organization (WHO), nearly 120 million units of blood are donated every year. However, this is not sufficient to meet the global need many patients requiring a transfusion do not have timely access to safe blood. Regular donations are required to ensure there is always a supply for those in need. Maintaining safe and effective procedures around the collection, storage and use of donated blood is essential. Collectively called haemovigilance, these procedures cover the entire blood transfusion chain and are used to standardize the use of blood in healthcare.

The Blood Transfusion Service (BTS) in India is fragmented and disintegrated under various controls, and there is a wide gap between demand and supply. As said by the Executive Director of Thalassaemia International Federation, India is a subcontinent with a population of 1,380,004,385 people (17.7 per cent of the total global population). It has an estimated prevalence of patients with Transfusion-Dependent β-Thalassaemia (TDT) of 150,000 with a predicted annual number of affected births of 12,500. With such a disease burden, there is a huge requirement for Blood centers and blood donors. Based on current estimates, an estimated two million units/year of packed red cells are needed to address the needs of TDT in India [58].


**Table 1.**

*Population screening & preventive measures needs to be taken.*

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Indian Ministry of Health and Family Welfare (MoH& FW) identified 2626 functional Blood centers across the country in the year 2016 from which, 76 per cent were public and not-for-profit-owned and 24 per cent were owned by the private sector. However, 61 per cent of these were situated in eight states, out of which only two (Maharashtra and Gujarat) have a high thalassemia prevalence. The Blood centers/ million population in high thalassemia-prevalence states, including Uttar Pradesh (1.2), West Bengal (1.3), Rajasthan (1.5) and Chhattisgarh (two) is less than the national average of 2.2 Blood centers/1,000,000 population. It was estimated in 2017 that the annual collection was 11.1 million units of blood while the demand was 14.6 million units [58].

In Thalassemia Major Patient's transfusion therapy is often initiated before one or two years of age [59–62]. Complications directly related to transfusion include blood-borne infections, development of anti-RBC antibodies (both auto- and alloimmunization), and allergic, febrile or delayed hemolytic transfusion reactions. Hb levels above 12.0 g/dL for adult women and 13.0 g/dL for adult men are considered normal [63]. General transfusion guidelines recommend initiating transfusions at an Hb threshold of 6.0–10.0 g/L, depending on the presence and severity of clinical conditions [64]; however, these guidelines focus mainly on correcting anemia rather than suppressing ineffective erythropoiesis and may not be applicable to patients with β- thalassemia [65]. Guidelines for the management of β-thalassemia are available, including international guidelines by the Thalassaemia International Federation (TIF) [66, 67] and several national guidelines [68–70].

#### **3.1 Guidelines for blood transfusion in Beta-thalassemia patients**

Life-long and regular blood transfusion is required to treat thalassemia major. This results in excessive accumulation of iron in the body (iron overload) that in the long term gives severe clinical complications such as heart and liver failure, diabetes, hypogonadism. Iron overload may be prevented and treated by daily removal (iron chelation therapy) [71–73].

Patients with β-thalassaemia major should receive leucoreduced packed red blood cells with a minimum hemoglobin content of 40 g [66, 67]. Only the transfusion of pRBCs can maintain the required hemoglobin percentage necessary for the normal growth of the diseased child. The transfused blood should be obtained from voluntary non-paid donors and collected, processed, screened, stored and transported by a trusted and high-quality blood transfusion centre [59]. If possible fresh blood should be transfused (not older than 10–12 days) as 2, 3-DPG (2, 3- Di-phophoglyceric acid) gets depleted in stored blood that reducing the capacity to deliver oxygen to the tissues [74]. Decreased recovery and shortened half-life may increase transfusion requirements. For all these, it is important to strengthen the Blood centers and component therapy should be ensured and mandatory. As per the guideline issued by the Indian Ministry of Health & Welfare in the year 2016 [28], A blood centre should be fully occupied with good infrastructure having component separation facility without that packed red cells are not available, and pretesting strategies should be followed for such chronically transfused patients are;


*Novel Diagnostic Approach and Safe Blood Transfusion Practices for Thalassemia… DOI: http://dx.doi.org/10.5772/intechopen.101672*


#### **3.2 Role of blood Centre in maintaining the national guidelines for β-thalassemia patients**

As mentioned above it's the huge responsibility of every blood center to provide safe blood to these chronically transfused patients. Flowing are the procedures that should be carried out at every blood centre for providing safe blood and managing such patients.

#### *3.2.1 Blood donations, quality of blood and Donor's motivation*

For the health of chronically transfused patients with thalassemia, a blood donor should be careful by keeping the criteria of regular voluntary and non-paid donors [26, 28, 79]. The process of donor selection is usually done through questionnaires. These questionnaires are prepared by considering the national need, resources, the prevalence of the transfusion-transmitted infections and also by adhering to the directions from the European Union (EU), World Health Organization (WHO), American Association of Blood centers (AABB). Quality of collected blood would be maintained by proper collection, testing, stored and distribution following all the quality-controlled procedures. The huge problem of low blood donation and blood supply is a lack of voluntary and non-paid donors. A public awareness campaign, including posters and an annual event to honor donors, was implemented to encourage voluntary and non-paid blood donors [80].

#### *3.2.2 Leukoreduced packed red blood cells*

Leucocyte-reduced red blood cells concentrate should be prepared by a method known to deplete leucocytes in the final component to less than 5x108 when intended to prevent febrile reactions and to less than 5x106 when it is required to prevent alloimmunization or CMV infection [26, 28, 71, 79, 81]. For achieving a level < 5x106 , the use of a leucocytes filter is necessary. Reduction of leukocytes to 5x106 is considered the critical threshold for eliminating adverse reactions attributed to contaminating white cells and for preventing platelet alloimmunization. Two types of preparation methods for Leucocyte depleted packed RBCs are;

• **Prestorage filtration**: is the process to remove white blood cells from the whole blood which is carried out with an in-line filter within 8 hours after blood collection. Using this technique very high-efficiency filtration is achieved with consistently low WBC residue and high red cell recovery. The removal of the WBCs before storage will prevent the accumulation of cytokines during storage that leads to a reduction in the number of FNHTRs. This leukoreduced whole blood is centrifuged to obtain packed red cells.

• **Bedside filtration**: This technique is used for filtering red cell concentrators (RCC) at the bedside which may not allow optimal quality control, as the techniques used for bedside filtration are highly variable. Red cell concentrates are prepared from stored whole blood by removing plasma and buffy coats.

#### *3.2.3 Other specific products for Thalassemic patients*


#### *3.2.4 Amount of blood to be transfused*

Packed red blood cells 15 ml/kg body weight, should be administered at the rate of 5 ml/kg/hr [28]. As per the pre-transfused hemoglobin level, 1–2 units of pRBCs may be required by patients. 3.5 ml/kg of pRBCs with around 60% HCT can raise hemoglobin by 1gm/dl.

#### *3.2.5 Storage and transport of red cell units*

Different anticoagulant solutions are used to store the blood products which prevent coagulation and store red cells without losing their metabolic activity [28, 71, 79, 81]. **Table 2** shows the list of anticoagulants that are commonly used. In thalassaemia major decreased recovery and a shortened red cell half-life may increase transfusion requirements and as a consequence the rate of transfusion iron loading, the current practice is to use red cells stored in additive solutions for less than two weeks. Blood units should preferably be transported in monitored insulated boxes which maintain a temperature of between 2 and 8°C.

*Novel Diagnostic Approach and Safe Blood Transfusion Practices for Thalassemia… DOI: http://dx.doi.org/10.5772/intechopen.101672*


**Table 2.**

*Anticoagulants and their shelf life period.*

The following data should be regularly recorded at each transfusion:


#### *3.2.6 Compatibility testing and alloantibody detection*

Every recipient should receive ABO & RhD type specific compatible whole blood or red blood cell components [26, 28, 71, 79, 81]. 'O' packed red cells should be transfused only when ABO type specific unit is absent. RhD positive recipients can receive either RhD positive or negative components. But RhD negative recipient should receive transfusion only of RhD donor; however, in reasonable circumstances, RhD positive unit may be transfused only when the receiver does not have the RhD antibodies. In the case where clinically significant atypical antibodies are detected, a negative unit particular for that antigen or least compatible unit should be transfused. When a patient is massively transfused within a period of 24 hrs, a fresh sample should be used for subsequent transfusions.

In the case of multitransfused patients like thalassemia major and sickle cell disease, blood transfusion should be done after the confirmed diagnosis. Before the first transfusion, patients should be typed for complete red cell genotyping that help to determine subsequent development allo-antibodies in such patients after repeated transfusion of different blood units. The development of multiple alloantibodies is common in these patients [88]. Therefore, it is important to monitor the patient carefully and to give him/her particular antigens negative blood unit for transfusion. Anti-E, anti-C and anti-Kell alloantibodies are most common. However, 5–10% of patients present with alloantibodies against rare erythrocyte antigens or with warm or cold antibodies of unidentified specificity [79].

Even before embarking on transfusion therapy, patients should have been gone through extended blood group antigen typing at least for C, c, E, e, and Kell which would be beneficial to identify antibodies in case of alloimmunization [79]. The rate of alloimmunization among transfusion-dependent thalassemia (TDT) patients is varying from 2.5–37% in different parts of the world [70, 87, 89]. The prevalence of anti-E, anti-c, and anti-c with anti-E in the Indian population is about 22–36%, 6.4%–38.8%, and 6.4%, respectively [90–92]. Relative immunogenicity of blood group antigen in a multi-transfused patient with an increase in a number of RBC unit exposure give the following grading: K > Jk<sup>a</sup> > Lua > E > P1 > c > M > Leb > C > Lea > Fya > S [93]. Extended blood group antigen phenotyping should be done to reduce the risk of alloimmunization. Keeping that thing in mind; our blood center phenotyped 500 'O' grouped donors for 35 blood group antigens [94].

#### *3.2.7 Adverse transfusion reactions*

Patients dependent on blood transfusion for survival, are exposed to a variety of risks [71, 79]. Therefore, it is utmost important to improve blood safety, find ways to minimize or reduce transfusion requirements and less exposures to number of donors. The adverse transfusion events are:


of the pro-inflammatory mediators during the storage of donor red cell [96, 97]. Management includes oxygen, administration of steroids and diuretics, and, when needed, assisted ventilation.

• **Transfusion-induced graft versus host disease (TI-GVHD):** caused by the viable lymphocytes in the donor's red cell unit, a rare but fatal complication due to transfusion. TI-GVHD may occur in immunosuppressed as well as immunocompetent patients. To avoid this, a blood transfusion of the family member's donated blood should be given after irradiation. Leucodepletion alone is inadequate for the prevention of this complication.

#### *3.2.8 Transfusion transmitted infections (TTIs)*

Blood safety depends on both donor health screening and donation testing. The strategy employed for a given infectious agent depends on the epidemiology of the particular agent in a given donor population, blood processing steps that might reduce transmission (such as pre-storage universal leucoreduction), and the availability of testing equipment and kits adapted for donor screening [98].

The different testing platforms can be used for screening TTIs. Tests that detect viral nucleic acid (NAT testing), a viral component (Hepatitis B surface antigen, or HBsAg; the p24 antigen of HIV), or the host's immune response to the infection (antibody testing performed using Enzyme linked immunosorbent assay (ELISA) or enzyme immunoassay (EIA)/chemiluminescence assay (CLIA) for antibodies such as anti-HIV, and anti-HBV). Any unit tested positive once for TTI must not be used for transfusion. Window period infections can be missed by ELSIA and chemiluminescence assay [98].

In general, window periods are shortest for NAT testing, longer for antigen testing, and longest for antibody detection. The lag time for anti-HIV to detect HIV infection can be as long as 21 days, for HBsAg test to detect HBV infection as long as 42 days, and for anti-HCV to detect HCV infection as long as 60 days. This lag period gets truncated (shortened) by direct tests for viral gene amplification with NAT [98]. Being costlier investigation than ELISA and chemiluminescence assay, NAT testing is still not available at many centers in developing countries like India [99].

Single unit NAT testing is more sensitive than using mini pool NAT where small numbers of donor samples are pooled [100]. Using this mini pool NAT of 10 samples, at our centre more than 8000 seronegative units were tested, out of those 44 donors were found positive for HBV, 5 for HIV and 2 for HCV. So ultimately these NAT positive units were stopped to be transfused to the patients [101]. Thalassemia and Sickle cell anemia patients should be transfused to the NAT tested unit [28]. In India only a few blood center hospital-based or private are doing either ID-NAT or mini-pooled NAT [99, 102–104]. In the year 2019 at our centre, as a part of the regular protocol, we tested 196 thalassemia major patients who are taking blood units from our centre only for transfusion, by ELISA and NAT (ID-NAT) for HIV1, HBV and HCV, the prevalence of HCV infection was found high by both the methods [105].

The testing algorithms for TTI s are also variable as per the testing facilities available at the blood centers. In the urban areas, screening for TTI is carried out by the ELISA technique with good quality measures while the remote rural areas are still left with a rapid test of questionable sensitivity. Moreover, in the remote rears where laboratory testing for TTI's is not quality assured, equipment is not calibrated and maintained, and the validation of results is not carried out. Even at the good centers that are doing ELISA using 3rd generation kits; not able to cut down the

window period which can be achieved by 4th generation ELSIA. It's a big pressure on the Indian Blood center to adopt NAT for screening TTIs as Blood centers around the countries like USA, Canada, Australia, New Zealand, South Africa, and some countries in Europe and Asia have already used the same [106].

Because of the need for sensitivity in testing systems, TTI screening may lead to false biological false positive results. Many TTI test systems rely on cut-off values to determine whether a result is reactive or not, so the test results that fall close to a range of uncertainty may give the intermediate result. For such kind of indecisive results, extreme care needs to be taken with follow-up action for that particular donor, including confirmatory testing on the donation. The confirmed reactivity to certain TTIs such as HIV, HBV and HCV leads to permanent exclusion of the donor, whereas the risk of other TTIs such as malaria may have specified time deferrals [98].

#### *3.2.9 Iron chelation and chelation*

On an average, each unit of packed cells contains 200 to 250 mg of iron [28, 79]. A patient, who receives 15–30 units of pRBC units per year, receives an excess of 3–6 grams of elemental iron that results in to iron overload, a serious problem in massively transfused patients. Iron supplements are contraindicated as iron absorption may increase up to 1–2 gm. Serum ferritin, MRI of Liver and heart, and Liver biopsy would be good parameters to check the severity of iron over load in multitransfused patients. Iron chelation therapy should be started in patients whose serum ferritin value is >1000 μg/L after 10–15 transfusion. Different chelating agents are mentioned in the guidelines issued by the Ministry of Health & Family Welfare, India in 2016 for the prevention of hemoglobinopathies. They are Desferrioxamine (recommended dose is 25 mg-50 mg/kg/day) [71], Deferiprone (standard dose is 50 mg–100 mg/kg/day), Deferasirox (administered at a dose of 20 mg–40 mg/kg/ day) and combination of desferrioxamine and deferiprone should be available for the patients. Before administering any chelating agent to the patient, the toxicity of an agent and required monitoring measures should be taken in account.

#### *3.2.10 Molecular genotyping*

Over a century, for detection of RBC antigen, a gold standard method, hemagglutination has been used to predict phenotype. Hemagglutination also term as serology is a sensitive, easy to perform, low costing and specific technique for the determination of RBC phenotype and is considered as optimal method for patient care. The serology technique reduces issuing time for blood units by extensive typing of donor's antigens. On other hand, with advances in immunohematology gives an understanding of the molecular basis of many blood group antigens. Molecular genotyping will help to type donors for a wider spectrum of minor blood group antigens and also genotype blood group antigens of multiply transfused patients such as sickle cell anemia or β-thalassemia or patients having positive direct antiglobulin test [106].

In multitransfused patients, haemagglutination fails to phenotype the patient's antigens due to donor-derived erythrocytes from previous transfusions. The molecular background of blood group polymorphisms is used for blood group antigen typing [107]. Previous studies have shown that molecular methods prove successful in determining the correct antigen profile of a multitransfused patient [108–110]. Both the blood donors and recipients can be genetically typed for all the clinically significant blood group antigens and antigen-matched blood can be provided to the recipient [108–112].

*Novel Diagnostic Approach and Safe Blood Transfusion Practices for Thalassemia… DOI: http://dx.doi.org/10.5772/intechopen.101672*

This approach could significantly reduce the rate of alloimmunization. Many PCR-based molecular detection assays are available. As per the laboratory facilities low throughput, medium- throughput and high-throughput PCR-based assays are available for blood group genotyping, PCR-RFLP technique though to be the first to be used for the purpose [107, 113, 114]. Many researchers around the world have used different PCR-based platform to genotype clinically significant antigens among multitransfused patients as well as in blood donors [115–123]. Our blood center has also typed regular voluntary blood donors, multitransfused patients (Thalassemia major & Sickle cell disease) and the tribal population [124, 125] for Rh, Kell, Duffy and Kidd blood group system; there was the statistically significant difference was observed in the phenotypic and genotypic prevalence of all these system's antigens especially in the thalassemia major patients.

#### **4. Conclusion and future recommendations**

Haemoglobinopathies in India are always the burning problem especially Beta-thalassemia major and Sickle cell disease; due to various aspects including screening of the patients in remote areas, lack of awareness about the severity of the diseases in rural as well as in urban areas, consanguine marriages, poor antenatal and prenatal screening tests, delay in timely detection of the diseased child, having misconceptions, myths, rigidity and superstitions among the general population, inert-caste marriages likewise many more adding to the burden.

SRKRC being the first charitable blood Centre in Surat city is providing safe blood for the last 44 years by following all the National guidelines. Just like what a blood centre should do, SRKRC is continuously updated with the current trends in transfusion medicine for the management of, especially multi transfused patients like Beta-thalassemia major and SCD. Starting from supplying perfectly crossed matched washed red cells or leuco-depleted pRBC's units to these patients. SRKRC has also implemented regular time interval strategies for irregular antibody screening, investigations TTI's, and providing the iron chelation therapy as well for batter management of these patients. Although for TTI's, ELISA is the mandatory test, in addition to that SRKRC is doing NAT testing which reduces the window period of TTI's and adds an extra layer of safety to the transfused blood units. Transfusion reactions are also one of the problems with these patients, antigen matched blood or antigen-negative blood (in case of alloantibody in-patient) should be transfused by typing patient and donor thoroughly with gold standard serology and advanced molecular genotyping techniques. SRKRC has developed a molecular genotyping facility for clinically significant blood group antigens that would serve the same purpose. As a blood centre, SRKRC is doing all above-mentioned possible transfusion practices and protocols that may help to enhance the time interval between two transfusions.

Along with routine blood centre activity, SRKRC is also running prevention and control programs for Beta-thalassemia and Sickle cell anemia for the last 16 years. This program includes the screening of the premarital, antenatal, PND and neonatal cases with proper counseling for every target population that significantly helps to prevent and control the disease.

Hence, it is suggested that nationalized policies are needed to be implemented at the micro-level for screening, counseling, prevention, blood transfusion and management of such patients to achieve the final goal of 'HAEMOGLOBINOPATHIES FREE COUNTRY'. India has many international, national and state-wise prevention and control programs for different diseases like HIV, Tuberculosis (TB), Malaria, SCD, etc., that include testing, medication and management of the individual

patient at very cost-effective or free of cost manner. Likewise, for not just Betathalassemia but also for transfusion-dependent and severe hemoglobinopathies, nationalized prevention and control programs should be formed by combining the screening, counseling, blood transfusion and treatment aspects.

National Medical Commission (NMC) of India is running a doctor of medicine (MD) PG programme on the subject of Transfusion Medicine (TM) in most medical colleges in India. It is a clinical MD degree after MBBS and recognized by NMC and the curriculum completely covered all the advanced and clinical aspects related to Immune hematology, Blood Transfusion therapy, Blood Centre Direction, haemoglobinopathies and its management. More than 150 TM specialist doctors passing out every year in India, so enough specialist doctors would be available to meet the requirement of the concept programme. A concept, "District Haemoglobinopathy Clinic" along with Regional Blood Centre, programme under the name of #Make Country Free from Haemoglobinopathies is to be recommended.

Aim, Facilities and goals of the "District Haemoglobinopathy Clinic" should be like;

#### **4.1 Diagnosis, Registry and Management of diagnosed patients**


#### **4.2 Watertight screening, counseling and prevention for diseases**


of 100% Newborn screening for haemoglobinopathies in a particular district and to enroll start the early quality care & medical treatment for the diagnosed newborn at District haemoglobinopathy clinic.

• 100% mandatory hemoglobinopathies screening report at pre-marital/adolescent age stage during the education and before marriage registration of couples, identifying the carrier state person, counseling and prevention for the same.

All these facilities at one place would be a great help to an affected child and a family that they should not have to roam here and there, although to set up such facilities Government aids are much needed.

These district-level clinics should be integrated inter-districts through Nodal officers to create State-level registry and database; these State-level reference clinics should be integrated Inter-State through Zone level officer of the Country to create National level registry and all databases. These could be assessable by caregiving physicians anywhere in the country, with all history and given therapeutic management, which could be helpful for better prospective care and increase the quality of life of patients. Only by implementing this level of concept programme, we could make Country Free from Haemoglobinopathies and zero birth of hemoglobinopathy case can be achieved in the Country.

#### **Author details**

Avani Shah, Sumit Bharadva\*, Parizad Patel and Kanchan Mishra Surat Raktadan Kendra and Research Centre, Surat, Gujarat, India

\*Address all correspondence to: sumit\_bharadva@hotmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 5
