Naked Eye Single Tube Red Cell Osmotic Fragility Test Research Articles

Prevalence and screening of hemoglobinopathies and glucose-6-phosphate dehydrogenase deficiency in Yemeni blood donors

ABSTRACT Hemoglobinopathies and Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency pose significant health threats in Yemen, which can be alleviated through robust preventive strategies, highlighting the importance of implementing a cost-effective and efficient blood donor screening method to facilitate early diagnosis and management of erythrocyte disorders, ultimately improving both donor safety and patient health outcomes. Objectives This study aimed to determine the prevalence of total blood cell abnormalities, hemoglobinopathies and G6PD deficiency and evaluate the efficacy of red blood cell (RBC) indices, mentzer index (MI) and naked-eye single tube red cell osmotic fragility (NESTROF) test as screening tools for diagnosis of β thalassemia trait among Yemeni blood donors. Methods A cross-sectional study was conducted with 106 volunteer blood donors who met the national standard criterion of blood donation. Various tests were performed, including complete blood count (CBC), serum ferritin, sickling test, G6PD assay, NESTROF test and high-performance liquid chromatography (HPLC). Results The prevalence of hematological abnormalities among blood donors reached 68.9%, with functional RBC abnormalities at 51.9%, leukopenia at 10.4%, thrombocytosis at 1.9%, and thrombocytopenia at 4.7%. Additionally, hemoglobinopathies were found in 21.7% of donors, with β-thalassemia trait at 3.8%, sickle cell trait at 1.9%, and suspected α-thalassemia trait at 16%, while G6PD deficiency and iron deficiency were present in 12.3% and 17.9% of donors, respectively. The NESTROF test, MCV and MCH demonstrated a sensitivity rate of 100%. MI and MCH exhibited the highest specificity followed by NESTROF test in the screening of β-thalassemia trait. Conclusions The prevalence of hemoglobinopathies and G6PD deficiency appear to be common among Yemeni blood donors. These results emphasize the necessity of comprehensive blood donation screening programs to safeguard the blood supply and promote early detection and management of hemoglobinopathies and G6PD deficiency in Yemen.

Assessing NESTROFT as a preliminary screening tool for thalassemia in the Malayali tribes of Dharmapuri district, Tamil Nadu, India.

Mutations affecting transcription, translation, or beta-globin stability, among other stages of beta-globin production, cause beta-thalassemia. Beta-thalassemia results in a high red blood cell count with a low mean corpuscular volume (60-70fl) and mean corpuscular hemoglobin (19-23pg), and Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT) is recommended for mass screening of populations. Among tribals in Dharmapuri district, this cross-sectional analysis evaluated the efficacy of RBC and NESTROFT against HPLC, regarded as the gold standard, in a study involving 484 subjects. Findings indicated that out of the 484 samples, 73 tested positive for the beta-thalassemia trait through HPLC. The NESTROFT test demonstrated 87% sensitivity, 98.5% specificity, a positive predictive value of 99.3%, and a negative predictive value of 73.3%. In the multivariate analysis, NESTROFT and hemoglobin showed high significance with P values of 0.003 and 0.000, respectively. NESTROFT's high sensitivity is particularly noteworthy due to the absence of research among the Malayali tribes in the Dharmapuri district. Adoption of this preliminary test shows promise in detecting the disease at the local level, providing important information for early detection, especially considering the sizeable tribal population.

Reproducibility of Naked Eye Single Tube Red cell Osmotic Fragility Test (NESTROFT) as a screening test for the detection of b thalassemia and Hemoglobin E traits with the variation of reading time

Background: Though 0.36% NaCl buffered solution has been used as an effective reagent for carrying out Naked Eye Single Tube Red cell Osmotic Fragility Test (NESTROFT) as a screening test for the detection of b thalassemia and Hemoglobin E traits, reading time for its interpretation is yet to be settled. Methods: This cross -sectional analytic study was done at Department of Pediatric Hematology and Oncology, BSMMU from January 2006-June 2007. NESTROFT was performed in 3 separate tubes for each of the 100 subjects (with â thalassemia trait and HbE trait and normal hemoglobin) with 0.36% NaCl buffered solution and reading was taken at different time at 5-minute, 15-minuteand 30-minute. Results were recorded as positive and negative. Hb electrophoresis, Hemogram with RBC indices for each sample and iron profile of selected subjects were done. Results: Among the 100 study subjects, 48 were carriers (18 were â thalassemia traits, 30 were Hb E traits). Age range was 1 to 52 yrs. Mean Hemoglobin concentration, MCV, MCH of both b thalassemia and Hb E carrier were significantly lower than normal subjects (p values <. 001). At 5-minute observation, NESTROFT was positive in 17 out of 18 â thalassemia traits with a sensitivity of 94.4%, 24 out of 30 HbE traits gave positive result with a sensitivity of 80%. Among the 52 normal subjects 35 gave negative result with a specificity of 67%. Positive predictive value and negative predictive value for detection of b thalassemia traits and HbE traits were 50% & 97% and 58% and 85%respectively. Result of NESTROFT interpreted at 15-minute and 30-minute did not show any change. Conclusion: From this study it may be concluded that for interpretation of NESTROFT reading at 5-minute would be optimum. BANGLADESH J CHILD HEALTH 2022; VOL 46 (3) : 130-137

Screening of Extended Family Members of Thalassemia Major Children as a Thalassemia Preventive Strategy.

Thalassemia is considered as the most common single gene disorder worldwide. Preventive measures include identification of thalassemia carriers (traits) through screening, genetic counselling and prenatal diagnosis to reduce the incidence. This study aims at estimating the prevalence of carrier status detection among the extended family members of children having thalassemia major so as to use it as a screening prevention strategy with appropriate counselling. This cross-sectional study was conducted in thalassemia unit of Pediatric Department of a tertiary care teaching hospital over a period of 18 months. Blood samples were collected from 117 extended family members (EFM) of 23 children with thalassemia major to carry out investigations such as Complete Blood Counts (CBC), Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT), Reticulocyte count, High Performance Liquid Chromatography(HPLC) and serum ferritin. Reports were analysed to find out the prevalence of carriers. Among 117 EFM, 62 (52.9%) were males while 55(47.1%) were females. Mean age distribution in this study was 16.49 years (8.5). Prevalence of thalassemia trait (carrier) was 35%. NESTROFT test was positive in 57(48.7%) participants. The binary logistic regression found only positive NESTROFT test as a predictor (adjusted OR=0.022, P=0.001) of having raised HbA2 (HbA2≥3.5 %). Screening of thalassemia carrier by targeting extended family members of thalassemia major children could yield more carrier cases and targeted counselling could help effectively in decreasing the number of children born with thalassemia major. This strategy could be included in future plan of national prevention programme for thalassemia.

Burden of Congenital Haemolytic Anaemias among the Rural Paediatric Population of Central India

Introduction: Childhood anaemia may be caused due to many factors, including malnutrition, chronic infections, deficiency of iron and vitamins, parasitic infections. On haematological work up the most common finding is microcytic hypochromic anaemia. Inherited defects in Red Blood Cell (RBC) and haemoglobin structure and metabolism result in congenital haemolytic anaemia. About 4.2% of all paediatric microcytic anaemias are diagnosed with haemoglobinopathies. Thalessaemia and other haemoglobinopathies have a large representation in the Indian subcontinent. A study of the extent of these hereditary anaemias will help in formulating a targeted approach of spreading awareness and educating couples in target population, who are planning conception. Aim: To comprehend the incidence of congenital haemolytic anaemias in the paediatric population of central India, and to evaluate their clinico-haematological status. Materials and Methods: This cross-sectional observational study was conducted in the Paediatric Anaemia Clinic at Chirayu Medical College and Hospital (teaching, tertiary care hospital), Bhopal, Madhya Pradesh, India, from January 2021 to December 2021. All relevant details regarding clinical history, consanguinity, family history of the patients were recorded. Investigations done included-complete blood count, reticulocyte count, sickling test, Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT) and High Performance Liquid Chromatography (HPLC). All the relevant data collected was entered into Microsoft Excel and frequency, distribution, standard deviation were analysed and calculated. Results: Amongst the total 1080 cases of microcytic hypochromic anaemia in the Paediatric Anaemia Clinic, there were 55 cases of congenital haemolytic anaemias. Of these 55 cases of congenital haemolytic anaemias, there were 41 (74.54%) cases of thalassaemia (four major and 37 minor thalassaemia), 11 (19.9%) cases of sickle cell anaemia, 1 (1.81%) case of Hereditary Persistence of Foetal Haemoglobin (HPFH) and 2 (3.63%) cases of Haemoglobin J variant (HbJ). The haemoglobin values were the lowest in the β-thalassaemia major patients, followed by sickle cell disease. Foetal Hb (HbF) was highest in β-thalassaemia major at and the HPFH case had foetal haemoglobin of 35%. Conclusion: In the current study, 5.09% of the paediatric population presenting to the clinic were diagnosed with congenital haemolytic anaemias, with major representation of β-thalassaemia, followed by sickle cell trait. This lifelong burden of haemolytic anaemias on the children and their families may be reduced by active counseling and awareness programs.

NESTROFT-A Cost-Effective Mass Screening Tool for the Detection of β-Thalassemia Carrier Status in Anemic Pregnant Women: A Step Toward Reducing the National Disease Burden.

Introduction β-Thalassemias are inherited hemoglobinopathies commonly encountered in practice. With chances of a promising cure being rare, the prevention of births with this disorder should assume priority, especially in low-resource countries. This can be achieved by the implementation of a mass screening program that is reliable and, at the same time, cost-effective. Objectives This study focuses on the utility of Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT) as a mass screening tool to detect thalassemia carriers. Hematological parameters that may predict carrier status were also evaluated. Materials and Methods Hemoglobin estimation was performed on all consented pregnant women. If the patient was found to have hemoglobin < 11 g/dL, the blood sample was subjected to other routine hematological tests along with peripheral smear examination. NESTROFT was performed using 0.36% saline solution. Confirmation was done using high-performance liquid chromatography (HPLC). Statistical Analysis Data obtained were tabulated using version 21 of the Statistical Package for Social Sciences. Means, standard deviations, and percentages were used to describe the sample. Chi-square test and Students’ t test were used to identify differences between the groups. Results Of 441 pregnant women enrolled, 206 were found to be anemic. Nineteen (9.2%) of the anemic pregnant women were detected to be carriers of hemoglobinopathies. Among the hematological parameters, mean red blood cell count and reticulocyte count were higher, while mean corpuscular hemoglobin concentration was lower in carriers. Also, carriers were more likely to present with microcytic hypochromic anemia. NESTROFT showed a sensitivity of 84.21%, specificity of 96.25%, a positive predictive value of 69.56%, and a negative predictive value of 98.36%. A false-positive result was seen in 3.74% of the tests, while a false negative result was seen in 15.78% of the tests. Conclusions NESTROFT (0.36%) can be used as a simple and cost-effective mass screening tool for the detection of carrier status. This should be followed by confirmation using HPLC or hemoglobin electrophoresis.

Evaluation of Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT) in Screening and Diagnosis of Beta Thalassaemia Trait in Pakistani Population

Background: Beta thalassaemia is an inherited disease that is a cause of continuous burden on the affected families and the society. The objective of the study was to evaluate the efficacy of Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT) for the detection of beta thalassaemia trait when used for the screening in Pakistani population. Methods: All participants requesting a complete blood count were included in the study. Informed consent was obtained. Naked Eye Single Tube Red Cell Osmotic Fragility Test was performed with freshly prepared 0.36% buffered saline. Turbidity in the tubes was an indication for individuals to be positive for beta thalassaemia trait. Hemoglobin electrophoresis was performed on all positive cases to confirm the sensitivity and the reliability of the screening test. Chi-Square test was used to test for any significant correlation between the different hematological parameters and other variables. Results: A total of 812 patients were included in the study from January 2017 to June 2017. The mean age of participants was 35.5 + 13.6 years. There were 351 (43.2%) male and 461 (56.7%) female participants. A 100% Naked Eye Single Tube Red Cell Osmotic Fragility Test sensitivity was obtained with 65 (8%) positive cases having HbA2 levels of more than 3.2% indicating their true positivity whereas, all 747 (92%) negative cases having HbA2 levels of less than 3.2% indicating their true negativity. Conclusion: NESTROFT was found and a rapid screening test for the identification of carriers of beta thalassaemia trait in our population.

Screening to Determine Prevalence of β-Thalassemia and Iron Deficiency Anemia Among Medical Students

According to the Thalassemia Federation of Pakistan, the mostly inherited disorder in Pakistan is β-thalassemia, which is characterized by a deficient, abnormal, or lack of β-globin chain synthesis and has a prevalence of 6%. The only method of controlling and preventing β-thalassemia is to increase awareness among students. This was an observational study using a random sampling technique. The Dow-Thalassemia awareness program recruited 915 medical students from the Dow Medical College (DMC) and Sindh Medical College (SMC) to voluntarily donate blood samples, which were analyzed by the naked eye single tube red cell osmotic fragility test (NESTROFT) and complete blood count and results were confirmed by high-performance liquid chromatography and analyzed using the NESTROFT. The samples were collected in 2012-2013. A total of 915 samples, out of these 390 samples, 390/915 (42.6%) samples were positive and complete blood count found 282 (72.3%) were positive for iron deficiency anemia. The remaining 108/390 (27.6%) were confirmed by high-performance liquid chromatography. Only 2.4 % subjects were positive for the β-thalassemia trait. Of 915 students, 57.4% of students were healthy, 39.2% had iron deficiency anemia, and 2.4% were carriers of the β-thalassemia trait. The overall prevalence of β-thalassemia was 38/915 (4.1%), which was lower than observed in previous studies. This study also demonstrated the NESTROFT can be used as a primary method of screening out healthy individuals, where approximately 50% require further screening for β-thalassemia

Utility of screening tools to differentiate beta thalassemia trait and iron-deficiency anemia - do they serve a purpose in blood donors?

BackgroundThe aim of this study was to estimate the prevalence of the beta thalassemia trait (BTT) and differentiate it from iron-deficiency anemia (IDA) among blood donors.MethodsA total of 1,000 samples from blood donors were subjected to complete hemogram with red blood cell indices. Further, Mentzer index (MI) was calculated for samples with mean corpuscular volume (MCV) below 80 fL and mean corpuscular hemoglobin (MCH) below 27 pg. Samples with Mentzer index <12 were subjected to naked-eye single-tube red cell osmotic fragility test (NESTROFT) followed by hemoglobin electrophoresis in positive cases. Serum ferritin was assessed in NESTROFT-negative cases.ResultsThe prevalence of BTT among blood donors was 3.7% and that of microcytosis among donors was 8.6%. The prevalence of BTT among microcytic donors was 41.8% while that among those with IDA was 11.6%. A value of MI <13 was highly sensitive in the diagnosis of BTT. MI >13 was found to have both high specificity and high sensitivity for diagnosing IDA.ConclusionA moderately high prevalence of BTT was observed among blood donors. Presently, no screening program is mandatory for screening of BTT among blood donors. Indices like MCV, MCH, and Mentzer Index were thus found to be effective to screen for BTT and IDA among blood donors, and NESTROFT was a cost-effective mass screening method to differentiate BTT and IDA.

An analysis of NESTROFT and Red cell indices in evaluating antenatal mothers for Beta Thalassemia trait

Background: The frequency of thalassemia trait is about 3% worldwide, while in developing countries like India, it is a major cause of burden on the health care system. Naked eye single tube red cell osmotic fragility test (NESTROFT) used in population screening for beta thalassemia trait. It is a simple, low cost, reliable and most suitable screening test for β BTT, with a sensitivity of 99.8% in regions with high prevalence rates.Material &amp;Methods: The present study was conducted in the Department of Pathology and included a total of 174 antenatal cases, attending the Out Patient Department of Obstetrics and Gynecology of a tertiary health care center in New Delhi between June 2011 to January 2012. The aim of the study was to screen mothers antenatally for early detection of BTT and to test the validity of NESTROFT in detection of thalassemic carriers of this area.Results: 174 pregnant women attending antenatal clinics were screened for detecting hemoglobinopathy with the help of NESTROFT, red cell indices, haemoglobin electrophoresis and HPLC It was seen that only four cases were BTT while ten were of microcytic anaemia and two had normal red cell indices.Conclusion: antenatal diagnosis of BTT is an important screening programme to reduce the burden of birth of children suffering from thalassemia and other hemoglobinopathies.Bangladesh Journal of Medical Science Vol.17(3) 2018 p.411-416

English

In India, There Are Around 20 Million Carrier Cases Of -Thalassaemia And Around 8000-10,000 Children Are Born Every Year With -Thalassaemia Major, Lokeshwar M.R. Shah N, Kanakia N. Et.Al 2006. The Birth Of A Thalassaemia Child Places Considerable Health And Economic Strain Not Only On The Affected Child And His/Her Family But Also On The Community. Since, Thalassaemia Is A Severe And Incurable Disease, Emphasis Has To Be Shifted From The Treatment Of An Affected Child To The Prevention Of Such Births In Future. Identifying Carriers For - Thalassaemia Patients, Thus Plays An Important Role In Preventing This, Verma IC Et.Al (1992). - Thalassaemia Trait Presents With Microcytic Hypochromic Blood Picture Similar To The Picture Of Iron Deficiency Anemia And Anemia Of Chronic Diseases. The Differentiation Between These Conditions Becomes Mandatory Which Usually Relies On The Measurement Of Serum Ferritin, Serum Iron, Total Iron Binding Capacity And Hb A2 Levels. These Diagnostic Methods Are Costly And Hence Constitute A Significant Burden On Public Health Economy Particularly In Developing Countries Like India. This Made The Way For Developing A Simple And Economic Screening Test For The Detection Of Carriers Of - Thalassemia. In 1988 Mehta BC Et Al Developed A Simple Cost Effective Screening Test For The Detection Of Cases Of -Thalassemia Trait Based On Reduced Osmotic Fragility. This Is Called NESTROFT (Naked Eye Single Tube Red Cell Osmotic Fragility Test). We Have Tried To Assess The Usefulness Of This Screening Test In Our Paediatric Cases Presenting With Anemia.

PRE-MARITAL SCREENING TESTS OF β-THALASSEMIA TRAIT IN DAKSHINA KANNADA POPULATION OF KARNATAKA

&lt;p class="Default"&gt;&lt;strong&gt;Objective: &lt;/strong&gt;β-Thalassemia is one of the familiar single gene disorders which passes from parents to offspring. The prevalence of β-thalassemia trait varies from 1-14% in different regions of India. Every year almost 9000 β-thalassemic major children are being born in the Indian sub-continent. In the present study, the prevalence of β-thalassemia trait was checked and some screening tests were performed to detect it among the Dakshina Kannada population of Karnataka.&lt;/p&gt;&lt;p class="Default"&gt;&lt;strong&gt;Methods: &lt;/strong&gt;A total of 800 youngsters were selected for the study, males being above 21 y and females above 18 y. Two ml of blood was drawn and collected in K2 EDTA bottles and complete hemogram was immediately checked. Samples which have Mean Corpuscular Volume (MCV)&amp;lt;80 fico litres(fl) were selected for the study. Five discriminant functions were calculated. NESTROFT (Naked Eye Single Tube Red Cell Osmotic Fragility Test) was performed in all the samples. The samples which show positive for NESTROFT and at least 2 discriminant functions were further checked for HbA2 level using cellulose acetate electrophoresis to confirm the β-thalassemia trait. A comparison was made with the normal samples which have MCV ˃ 80fl.&lt;strong&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results: &lt;/strong&gt;Prevalence of β-thalassemia trait was found to be 5.125 % in this population. The obtained values were analyzed using unpaired Student’s‘t’ test using GraphPad prism (Version-3.0). Samples of β-thalassemia trait have significant changes in the white blood corpuscles (WBC p=0.1266), red blood corpuscles (RBC p=0.0130), hemoglobin (Hb p&amp;lt;0.0001), hematocrit (HCT p&amp;lt;0.0001), MCV(p&amp;lt;0.0001), mean corpuscular hemoglobin (MCH p&amp;lt;0.0001), mean corpuscular hemoglobin concentration (MCHC p&amp;lt;0.0001), platelets (PLT p=0.0005), HbA2(p&amp;lt;0.0001) compared to normal controls.&lt;strong&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusion: &lt;/strong&gt;The present study shows that the people with β-thalassemia trait have a significant variation in complete hemogram compared to normal; NESTROFT and discriminant functions can be used for the screening of β-thalassemia trait in the population.&lt;/p&gt;

A study of thalassemia screening of 1000 medical students and comparison of various screening methods

Background: β-thalassemias are widespread throughout many countries including India. India has 3.5 crores thalassemic carriers with about 10,000 thalassemic birth every year in India. In Gujarat, there are about 6000 thalassemic children. The prevalence of β-thalassemia trait (BTT) is not uncommon in India and its incidence is rising in certain states including Gujarat. Objective: (1) To evaluate the prevalence of BTT among medical students above 18 years in C.U. Shah medical college; (2) To compare specificity of NESTROFT (Naked Eye Single Tube Red Cell Osmotic Fragility Test) with Hb-Electrophoresis method for screening of BTT; (3) To compare specificity of Mentzer’s index with Hb-Electrophoresis method for screening of BTT; (4) To create awareness among medicos about BTT. Materials and Methods: Blood (2 mL) was collected from 1000 medical students; complete blood count (CBC), peripheral smear examination, and NESTROFT were done on all samples. HbA2 levels were measured by fully automated cellulose acetate electrophoresis GENIOS-INTERLAB on suspected samples for BTT on basis of CBC, peripheral smear findings, and NESTROFT. HbA2 levels >3.5 were taken as gold standard for diagnosing BTT. Results: In this study, we found the incidence of BTT being 4.1% (41/1000), that of iron deficiency anemia being 7.3% (73/1000) and that of megaloblastic anemia being 5.4% (54/1000). Three students were found to be sickle cell trait and one to be Hb D. These four cases were confirmed by HPLC at higher center. Conclusion: Prevalence of BTT is high in India and aggressive control measures should be taken to prevent it. Government and NGOs should concentrate attention to educate MEDICOS and public at large. This will decrease financial burden on families of thalassemia major patients and society at large.

The Use of NESTROFT for Screening Pregnant Women for Detection of β-Thalassemia Carriers

The thalassemias and sickle cell disorders are widely distributed in India and cause a significant health burden [1, 2]. Community control programmes are the only way to reduce this burden. Screening for identification of carriers forms an integral component of a prevention programme [1, 2]. The often-debated questions are whom to screen and which would be the most appropriate technology to be used. The individuals targeted for screening in most studies include school or university students, newly-married couples, pregnant women, extended family members of a thalassemia major child, or members of communities where the prevalence of b-thalassemia carriers is higher than the average of 3 %–4 % in the Indian population. Pregnant women would be the most appropriate group as they are the ones at immediate risk but they often come late in the second trimester of pregnancy to the antenatal clinic for their first visit, especially in public hospitals, and it is often difficult to get their husbands for testing [3]. All these target groups have been screened in India by different centers [4–7]. Premarital screening is largely not acceptable due to fear of stigmatization. The technologies used for screening and diagnosis, include the following: Naked eye single tube red cell osmotic fragility test (NESTROFT); red cell indices; discriminant functions; flow cytometric osmotic fragility; and hemoglobin analysis by high-performance liquid chromatography (HPLC), capillary electrophoresis, or cellulose acetate electrophoresis for estimation of Hb A2, Hb F or other hemoglobin variants. A combination of these methods has been used by different investigators and each one has its advantages and limitations [8–13]. NESTROFT has been used as a primary screening test for population screening in many studies after Kattamis et al. first showed that a 0.36 % saline solution was sensitive and effectively detected more than 96 % of b-thalassemia carriers on screening [14]. The ideal approach would be to measure the red cell indices and quantitate Hb A2, Hb F, and any other hemoglobin variant by HPLC or capillary electrophoresis in every individual to ensure that all b-thalassemia heterozygotes as well as carriers of abnormal hemoglobins are identified. However, this is not a cost-effective strategy for massscreening programmes in a large country. Moreover, even with this ideal strategy, around 2 % of b-thalassemia heterozygotes who have normal red cell indices, and/or normal or borderline Hb A2 levels will be missed. NESTROFT has a high sensitivity but lower specificity as shown in several studies [8–13]. Using NESTROFT as a single first-line screening test has some limitations. Although it has a high negative predictive value, few bthalassemia carriers have been missed in different studies. In a quality control evaluation done during the large multicenter study of Indian Council of Medical Research (ICMR) under the Jai Vigyan programme, the main reasons for higher number of false negatives at some centers were the quality of water used, inaccurate dilution of the buffer, and frequent change of technicians. It was also shown that if NESTROFT was combined with measuring red cell indices in the first step itself using mean corpuscular volume (MCV) \80 fL and mean corpuscular hemoglobin (MHC)\27 pg as cut-off values, the b-thalassemia carriers & Roshan B. Colah colahrb@gmail.com

Hemoglobinopathy screening by osmotic fragility test based on flow cytometer or naked eye.

Diagnosis of hemoglobin (Hb) disorders is based mostly on abnormal red blood cell (RBC) indices, elevated levels of HbA2, HbF, or any other Hb on the Variant high performance liquid chromatography (HPLC) system, and confirmation by molecular methods. However, large scale population screening is of prime importance and requires a simple, accurate, and cost effective technique. We have tried to compare the sensitivity of the widely used Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT) and the osmotic fragility described as % residual RBCs through flow cytometry for population screening. The count of residual red cells was measured sequentially in real-time using flow cytometry. NESTROFT was performed using a 0.36% buffered saline. HbA2 and HbF levels along with other abnormal Hbs were determined on the Variant HPLC System. Molecular studies were done to confirm the diagnosis. The normal group showed a significantly lower percentage of residual RBCs (48.08 ± 11.87) as compared to cases (β thalassemia trait-82.97 ± 12.20, α thalassemia trait-72.58 ± 8.34, and HbS trait-85.00 ± 4.05). The sensitivity and specificity of NESTROFT was high for both β thalassemia traits (98.33 and 96.72%, respectively) and α thalassemia traits (100 and 96.72%, respectively) but very low sensitivity for HbS traits (54.84%). Flow cytometric osmotic fragility was a more sensitive method to discriminate normal from the group of hemoglobinopathy carriers as compared to NESTROFT which missed majority of HbS carriers. However, in view of feasibility and cost effectiveness, NESTROFT could still be used for population screening of thalassemia. © 2014 International Clinical Cytometry Society.

Incidence of the Hb E [β26(B8)Glu→Lys, GAG>AAG] Variant In Totos, One of the Smallest Primitive Tribes in the World

Toto is one of the smallest tribes in the world. This primitive sub Himalayan, endogamous tribe lives in a small, isolated village called Totopara in the Jalpaiguri district of West Bengal in India. The tribal communities of West Bengal are vulnerable to various genetic disorders such as β-thalassemia (β-thal). We have studied 443 Totos to define their Hb E [β26(B8)Glu→Lys, GAG>AAG] status. Awareness and screening camps have been organized in various parts of Totopara during the last 2 years. We collected 3 mL peripheral blood from each individual aseptically on which to use the naked eye single tube red cell osmotic fragility test (NESTROFT); complete hemogram and high performance liquid chromatography (HPLC) were done to detect their carrier status. The Hb E variant had been found to be prevalent among the Totos. To confirm the codon 26 (GAG>AAG) mutation in the β-globin gene, amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was performed. Restriction fragment length polymorphism (RFLP)-PCR was carried out with 44 Hb E alleles to construct the haplotype(s) of the Totos. Our extensive studies have revealed that 49.21% of Totos are Hb E heterozygotes and 19.19% Totos are Hb E homozygotes. The most prevalent haplotype linked with the codon 26 mutation in the Totos is [+ − − − − −] (HincII 5′ϵ, HindIII Gγ, HindIII Aγ, HincII 5′ψβ, HincII 3′ψβ and HinfI 3′β). Consanguineous marriages have resulted in a significant increase of the percentages of heterozygotes and homozygotes of Hb E in the Totos. Genetic counseling is essential and important to prevent the spread of this mutation and hence to save them from having any kind of clinically significant hemoglobinopathy in the future.

Prevalence of β-Thalassemia and Hemoglobin E in Two Migrant Populations of Manipur, North East India

Hemoglobinopathies are a group of inherited single-gene disorders found commonly in the Mediterranean region, Middle East, Indian subcontinent, Burma, and South East Asia. As many as 229 mutation variants of β-thalassemia are reported in the world, many community/region-specific mutations. Very little on thalassemia has been reported in Manipur, a North Eastern Indian state. Thus, a community genetic approach through population screening can be applied to the people of Manipur and a total of 602 blood samples from unrelated Meitei Brahmins (n=300) and Meitei Muslims (n=302) were screened for abnormal hemoglobins by Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT). HBA(2) level was estimated by using high-performance liquid chromatography and molecular analysis was done by using PCR. Sequencing was also carried out to confirm the mutations. High frequencies of NESTROFT positive (10%) and NESTROFT doubtful (11%) cases were found among the Meitei Brahmins in comparison to the Meitei Muslims [NESTROFT positive (3.31%) and NESTROFT doubtful (1.33%)]. The allele frequency of HB*E was 0.022 and 0.012 among the Meitei Brahmin and Meitei Muslim, respectively. Sequencing of the β-globin gene confirmed a very rare β-thalassemia [-90 C→T (0.33%)] among the Meitei Brahmins of Manipur. This mutation with hemoglobin E in one of the present population hints the presence of the Mongoloid stock among them, possibly from China. There is a need for further studies on other populations of Manipur and the neighboring North East Indian states using such genetic markers.

Validity of Naked Eye Single Tube Red Cell Osmotic Fragility Test for the Diagnosis of β-Thalassemia –A Case Control Study

This study was conducted at the department of paediatrics, Rajshahi Medical College Hospital over a period of 2 years to see the sensitivity, specificity, positive predictive value and negative predictive value of Naked Eye Single Tube Red cell Osmotic Fragility (NESTROF) test for the diagnosis of β-thalassemia. Fifty eight children with β- thalassemia were taken as case and 58 children were taken as control. Among the control 29 children were normal (without anaemia) and 29 children had anaemia other than β-thalassemia. Hemoglobin electrophoresis was done to all children to enroll them as case or control. Then NESTROF test was performed in all cases and controls. The data was analyzed by SPSS V.12. This study showed that sensitivity, specificity, positive and negative predictive value of NESTROF test was 96.55%, 72.41%, 77.77% and 95.45% respectively.&#x0D; TAJ 2011; 24(1): 1-5

Prevalence of Sickle Cell Anemia In Eastern India

AbstractAbstract 4822 Background:Sickle cell disease is an inherited disorder of hemoglobin synthesis. This is due to replacement of Valine for Glutamic Acid in position six of the Beta globin chain of hemoglobin. This genetic alteration yields unstable RBC which lasts for 10–20 days. In stressful conditions the cells become sickle shaped and get lysed. There are about 20 million people with sickle cell disease in India. During January 2009- May2010 camps were held in various parts of West Bengal, Jharkhand, Chattisgarh. Along with various mutations of thalassemia, we also observed sickle cell anemia among them. This triggered our interest to study the spectrum of the sickle mutation co-inheritant with different mutations such as Homozygous Sickle Cell, Sickle Cell-Beta0 Thalassemia, Sickle Cell-Beta+ Thalassemia, Severe β+ thalassemia genes, Moderate β+ thalassemia genes, Mild β+ thalassemia genes Sickle cell-HbE Thalassaemia, Sickle cell-HPFH Thalassaemia, in said part of India. Since Indian patients with SS disease had higher hemoglobin, red cell counts and higher HbF levels and lower HbA2, MCHC, MCV, and reticulocyte counts, hence a high hemoglobin is a risk factor for painful crises and may also be a risk factor for avascular necrosis of the femoral head, proliferative sickle retinopathy, and acute chest syndrome. Methods:We have screened 332 individuals in eastern part of India during the period January 2009- May 2010. 3ml of peripheral blood was collected in EDTA vial from each individual. NESTROFT (Naked Eye Single Tube Red Cell Osmotic Fragility Test) was performed on spot. Then Complete Blood Count was done within 24 hours of collection. HPLC (High Performance Liquid Chromatography) was performed to identify the samples for confirmation. In our observation in case of sickle cell anaemia HbF (Fetal haemoglobin), Hb (haemoglobin), MCV (mean corpuscular volume) values ranges between 0–10 %, ≤7-10g/dl, 65–90fl respectively. ARMS (Amplification Refractory Mutation System) PCR (polymerase chain reaction) was done to confirm the mutation. Result:LocationTotal no. of IndividualNumber of carrier with mutationNumber of patient with mutationJharkand1032917Chattisgarh115279West Bengal114317 Conclusion:Of the total samples collected in the camps held at various places of Jharkand, Chattisgarh & West Bengal 87 of them was carriers of sickle cell anemia. There was 7 homozygous (SS), 14 sickle beta, 12 double heterozygous for HPFH (High Persistance of Fetal Hemoglobin) & sickle cell anemia. In conclusion, the manifestations of sickle cell disease are influenced by a variety of other genetic and environmental factors. The occurrence of the disease against different genetic and environmental backgrounds provides experimental models that contribute to understanding the variability in clinical and hematological expression of the disease. Disclosures:No relevant conflicts of interest to declare.

Prevalence of Thalassemia Among Rabhas; a Small Tribe in Eastern Part of India.

Abstract 5114 BACKGROUNDThalassemia syndromes are a heterogeneous anemia caused by mutation affecting globin chains of the hemoglobin molecule. It is estimated that the average life span of Rabhas (tribal population of North Bengal, Jalpaiguri) range from 42 – 45 years. This raised a concern that they are perishing due to some genetic disease. Hence Screening Camps were conducted to identify the reason. Rabha is a little known Scheduled Tribe community of West Bengal. Assam now renamed to Asom is a North-East India state of India with its capital at Dispur. The Rabha people are mainly found in the jungles of Jalpaiguri. West Bengal is a States and territories of India in eastern India. With Bangladesh, which lies on its eastern border the state forms the ethno-linguistic region of Bengal. Jalpaiguri is the largest district of North Bengal, covering an area 6,245 sq.km. It is situated between 26 16' and 27 0' North latitudes and 88 4' and 89 53' East longitudes and Cooch Behar district. Cooch Behar is a district of the state of West Bengal, India, as well as the name of the town which gives its name to the district. The whole area of Eastern and Western Dooars, may be termed as the cradle land of the Rabhas. The Rabhas belong to Indo-Mongoloid group of people and have similarities with other members of Bodo. Bodo may stand for: *Bod A city in Norway* An ethnic community in India: the Bodo people*The Bodo language spoken by them. group such as Garo (tribe). The Garos are a tribe in Meghalaya, India, and Mymensingh District, Bangladesh, who call themselves Achik. Kachari. The Kacharis are the most widely spread tribe in northeast India. They are said to be the earliest inhabitants of the Brahmaputra Valley. Mech (tribe), Koch. Our objectives were- Awareness among the tribal population through talks and documentaries and discussions with community leaders with demonstrations at village level. The goal of thalassemia screening is to identify the carrier status among Rabha populations, to control the birth of affected children thus eradicating thalassemia among them & to save one of the oldest tribe from being extinct. MATERIAL & METHODSAt first an Awareness Programme was held among the Rabhas & then with the written consent peripheral blood was collected for thalassemia screening test. The screening age lies between 10 – 35 years. Firstly, NESTROFT (Naked Eye Single Tube Red Cell Osmotic fragility Test) was performed for spot detection. This was followed by CBC (Complete Blood Count) & HPLC (High performance Liquid Chromatography) for confirmation.Molecular Analysis of every sample was done using ARMS PCR.All together 277 individuals were screened. Of which 119 (43%) were HbE carrier & 110 (40%) were HbE homozygous. Rest of them was normal. The carrier & homozygous status was confirmed by performing ARMS PCR. The sensitivity of NESTROFT in this case was 95 %. CONCLUSIONThus the percentage of HbE carrier & HbE homozygous is very high among the Rabhas. But one thing is to be noted that their % of haemoglobin is very high. This might be one of the reasons that they do not require blood transfusion during their life span. It is spreading like a rapid fire due to consanguineous marriage among them. This is one of the reasons for the early mortality. In our project of Department of Biotechnology, Govt. of India we'll complete carrier status detection of total Rabha population (11,000) within 3 years. DisclosuresNo relevant conflicts of interest to declare.