Diagnosis of Hemoglobinopathies and thalassemias using both Haemoglobin capillary zone electrophoresis and High performance liquid chromatography - An experience from a tertiary care hospital in Northern India

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It has has been estimated that in India, 0.37 per 1,000 fetuses have Hb disorder and more than 10,000 children are born annually with severe hemoglobinopathy and only around 10.0% are managed optimally, therefore screening and diagnosis of carriers is of paramount importance especially in an antenatal OPD. While Haemoglobin capillary zone electrophoresis (HbCZE) and High performance liquid chromatography (HPLC) are commonly used methods for diagnosis of these entities, there are far and few studies which attempt to compare these two techniques. Haemoglobin A2 (HbA2) constitutes less than 3% of the total hemoglobin (Hb) in adults and the determination of Hb A2 levels is important to diagnose the beta thalassemia trait (BTT). In some cases, the level of HbA2 is not typically elevated and some difficulties may arise in making the diagnosis and hence accurate assessment of HbA2 becomes of paramount importance. HPLC and HbCZE are considered acceptable methods to diagnose BTT but these vary in their accuracy and cut-offs. Complex elution patterns as well as co-elution of variants like HbA2, HbE, Hb Lepore, HbD Iran create difficulty in interpretation of HPLC plots.The aim of this study was to compare the results of HPLC and HbCZE technique for screening of clinically significant haemoglobin disorders. On comparing the HbA2 values the HPLC showed higher values for HbA2 with a median values as compared to HbCZE while when no variant was present, the Hb A2 concentrations showed excellent agreement. The Hb F, HbS measurement agreement was good between both methods. To conclude HPLC and HbCZE are complementary techniques and normal ranges and means normal values differ between different methods and different manufacturers and therefore it is important that each lab establish its own cut offs. Haemoglobinopathies Haemoglobin capillary zone electrophoresis high-performance liquid chromatography Figures Figure 1 Figure 2 Introduction Sickle cell disease and thalassemia are the most common clinically significant haemoglobinopathies and the carriers of these disorders represent approximately 5.0% of the world’s population.The thalassemias and hemoglobinopathies are commonly encountered in areas where malaria is endemic because abnormal genes offer protection against malaria. They are classified as per the type of mutation (insertion-deletion, base change) or the affected globin chain (alpha chain, beta chain) structural haemoglobin variants including those involved in sickle cell disease, haemoglobin C (HbC) disease, and haemoglobin E (HbE) disease being the commonest Over 300,000 infants with severe Hb disorders are born each year(1-3). Thalassemia and hemoglobinopathy are the major health concern in the Indian subcontinent as the prevalence rate of beta thalassemia mutations is as high as 17% in some populations. (4) Hb E-beta thalassemia and sickle-cell anemia are also common with high incidence in tribal populations. The detection and characterization of a haemoglobinopathy involves a 3 tier work up. (1) Full blood count (2)Special haematological tests (3)Molecular testing.The value of routine haematological analysis along with morphology and red cell indices is a must for the part of work up in any hemoglobinopathy. Reticulocyte counts, HbH preparations, Heinz body preparations, solubility test for HbS, unstable haemoglobin testing using Isopropanol or heat stability test, Dichlorophenol Indophenol test (DCIP) for HbE screening, spectroscopic tests for unstable haemoglobins causing methemoglobinemia, P50 determination for oxygen (i.e. Oxygen affinity test), oxygen dissociation curves, etc are important tests to be performed in the testing for haemoglobinopathies.Most centres in India reporting the carrier frequencies of beta thalassemia and other haemoglobinopathies now use CBC and HPLC analysis of haemoglobins. Other methods include HbCZE, immunodiffusion (RID) and IFE (Isoelectric focusing).Molecular diagnosis of thalassemia is mainly applied for confirmation of screening results, for clarification of complicated cases, and or prenatal diagnosis. It is recommended that all positive screening results are confirmed through DNA analysis, and results should be interpreted altogether, including the evaluation of haematology and family history. Guidelines as per the ICSH for hemoglobinopathy screening not only recommend the use of quantitative techniques for thalassemia detection, they also stress the use of a confirmatory method when a Hb variant is detected. In an ideal setting, both techniques can be met by a combination of HPLC and HbCZE. Materials And Methods From March 2019 to December 2020, data from 536 whole blood samples sent to our institute for physician-ordered hemoglobinopathy screening were studied. The samples included patients being evaluated for microcytic hypochromic anaemia. Suspected iron deficiency anaemia unresponsive to iron therapy, prenatal/ antenatal screening, or sickle cell disease treatment and monitoring. High-Pressure Liquid Chromatography (HPLC) was performed using Bio-Rad Variant II β-thalassemia Short Program (Bio-Rad, Hercules, CA), while Haemoglobin capillary zone electrophoresis (HBCZE) was performed using Mini Flex Piercing analyzer( Sebia trivitron). Cases of human Immunodeficiency virus infection (HIV) on highly active human antiretroviral therapy (HAART), bone marrow failure syndromes (BMFS) Myelodysplastic syndromes (MDS) were excluded.A signed consent form was obtained from all the patients included in the study. Detailed clinical history and family history were obtained from each patient. Past history of blood transfusion, if present, was noted.Blood samples were collected in ethylene diamine tetrachloride acetate (EDTA) vials. For each patient, a peripheral blood smear (PBS) was prepared and stained with Leishman stain and observed for morphology to supporting the diagnosis of hemoglobinopathies. Hb variant analysis was performed for various hemoglobinopathies and variants using HPLC and HBCZE. Confirmatory tests such as acid and sickling test, solubility test, and brilliant cresyl blue test for HbH inclusions were performed as and when required. High-Pressure Liquid Chromatography HPLC analysis was performed using the manufacturer's instructions for the Bio-Rad Variant II β-thalassemia Short Program (Bio-Rad, Hercules, CA), which separates haemoglobin variants by cation exchange chromatography using a salt gradient. The VARIANT II is a fully automated HPLC system that can be used to separate and determine area percentages for haemoglobins A2 and F and to provide qualitative determinations of abnormal haemoglobins. The identification of these haemoglobin variants is made using retention time windows such as a "D-Window," "S-Window," and "C-Window." In the VARIANT II β-thalassemia Short Program the samples are automatically mixed and diluted on the VARIANT II Sampling Station (VSS) and injected into the analytical cartridge. Different haemoglobins vary in their retention times i.e. the time from application to elution from the column, their characteristic. Each sample run takes about six and half minutes and nine samples may be run in one hour. (5,7). Haemoglobin capillary zone electrophoresis Haemoglobin electrophoresis is in many ways an intermediary type of technique between classical zone electrophoresis and liquid chromatography which separates analytes according to the pH of the electrolyte solution and electroosmotic flow. For this study the capillary Mini Flex Piercing analyzer, Sebia trivitron was used. This instrument uses the principle of capillary electrophoresis in a free solution. The instrument is equipped to resuspend, lyse, separate, and analyze whole blood samples for variants collected in EDTA tubes. The haemoglobin bands were detected by absorption photometry and optical density measurements and converted to a migration image. This is then displayed as an electropherogram. The migration position for each haemoglobin in each sample is standardized relative to the position of the haemoglobin A and A2 bands and is measured in arbitrary units between 0 and 300. The distinct peaks in the migration image are assigned to one of 15 zones and are quantified as percentage haemoglobins A, F, A2 and C are provisionally identified and colour coded. 28 samples can be processed in one go and eight samples are run per hour. (6,7) Results A total of 536 patients with suspected haemoglobinopathy were studied. The age group of the study population ranged from 0.2 to 73 years with a mean age of 26.43 years. There were more females in the study group 377 females (70.34%) and 159 males (29.66%) owing to a large number of antenatal cases that were screened. The most common subset was patients referred for antenatal checkups 224(41.79%). Clinically pallor had been documented in 84 (15.67%) patients and 112(20.90%) cases had been sent as family screening cases.17 cases out of 536 (3.17%) presented had jaundice on presentation while a history of transfusion was documented in 53 cases. Out of a total of 536 patients that were screened 141 patients were detected to have underlying thalassemia or haemoglobinopathy. The majority of these patients were BTT 97(68.79%) followed by compound heterozygous Hb E -beta-thalassemia 15(10.64%) and heterozygous E state 12(8.51%). There were 02 (1.42%) homozygous beta-thalassemia patients and 03 (2.13%) each with Homozygous HbS and Heterozygous sickle cell state. One each (0.71%) of compound heterozygous HbS beta-thalassemia, compound heterozygous delta beta-thalassemia, HbD Iran, Heterozygous HbO-Indonesia, Haemoglobin G Sriraj /O Arab were also identified. Three samples had a previous history of transfusion and 01 sample showed a carryover of HbD of 2.7%. A repeat HPLC/HbCZE was advised for these patients but they were lost to follow up. The distribution of subgroups is depicted in table 1. The erythrocyte indices of the commonest subgroups are shown in table 2. Table 01:-Distribution of subgroups of study subjects Subgroups Frequency Percentage Homozygous beta thalassemia 2 1.42% Heterozygous beta thalassemia 97 68.79% Heterozygous E state 12 8.51% Compound heterozygous Hb E -beta-thalassemia 15 10.64% Homozygous HbS 3 2.13% Heterozygous sickle cell state 3 2.13% Compound heterozygous HbS beta-thalassemia 1 0.71% Compound heterozygous delta beta /beta-thalassemia 1 0.71% HbD Iran 1 0.71% Heterozygous HbO-Indonesia 1 0.71% Haemoglobin G Sriraj / O Arab 1 0.71% Post transfusion sample /carry over samples 4 2.84% Total 141 100.00% Table 2. Erythrocyte indices of the commonest subgroups heterozygous beta-thalassemia, Compound heterozygous Hb E beta-thalassemia, Heterozygous E state Erythrocyte indices Heterozygous beta thalassemia(n=97) Compound heterozygous Hb E beta-thalassemia(n=15) Heterozygous E state(n=12) Haemoglobin(g/dL) 11.3 ± 2.14 8.13 ± 3.46 11.61 ± 1.63 TRBC(x10 12 /l ) 5.57 ± 1.11 4.52 ± 1.15 4.62 ± 1.1 HCT 36.62 ± 6.32 29.11 ± 11.02. 35.93 ± 4.5 Mean corpuscular haemoglobin (fl) 55.3 ±85.3 61.32 ±8.32 73.79 ±4.38 Mean corpuscular haemoglobin (pg) 20.67 ±3.21 17.83 ± 3.7 23.81 ± 1.97 Mean corpuscular haemoglobin concentration (g/dL) 32.67 ± 2 28.79 ± 2.6 31.48 ± 2.39 On HbCZE the haemoglobin A values in the study group ranged from 3.2-99% with a mean HbA of 93.74 ± 1.Haemoglobin A2 (HbA2) constitutes less than 3% of the total haemoglobin (Hb) in adults and has almost no physiological importance however the determination of Hb A2 levels is important to diagnose the beta thalassemia trait (BTT).The haemoglobin A2 values in the study group ranged from 3.2-99% with a mean HbA of 93.74 ± 12.66 %. For the sake of statistical analysis HbA2 values were divided into the following groups for ease of comparison 7% with HbA2 < 2% indicative of IDA, alpha thalassemia, HbH disease δβ thalassaemia (if haemoglobin F was also elevated), 2.0 - 3.3% was essentially the normal values for HbA2, HbA2 of 3.4-3.7% was indicative of a borderline elevated HbA2, or severe iron deficiency in β thalassaemia trait. β thalassaemia trait with additional δ chain variant may also have lower values of HbA2. In the presence of haemoglobin S, HbA2 values are raised on HPLC and may fall in this borderline zone. Haemoglobin A2 values of >7.0% are rare and a structural variant needs exclusion after repeating HbA2 estimation. The majority of the patients 340 (63.43%) had levels of HbA2 within the normal range i.e. 2.0-3.3 % while the second most commonest was HbA2 levels of 3.8-7.0% totalling to 149 pts (27.80%). Six pts had borderline values of between 3.4 and 3.7% only two pts (0.37%) had values over 7%. On HPLC 352 (65.67%) pts had normal HbA2 values, and 136(25.37%) pts had HbA2 which fell into the range of 3.8-7.0%. 31 pts had values that were more than 7% and these were later sub-grouped based on the percentage of HbA2 present as Hb Lepore, HbE, and HbD Iran coeluate in this window. Values of HbA2 between 10-14% are indicated of Hb Lepore, 25-40% Hb E heterozygous, 44-48% HbD Iran and 70-90 % E homozygous. While the P2 window has the glycated Hb eluting in it, in the P3 window values up to 6% are acceptable 6-12% are indicative of sample deterioration and 15-25% are indicative of Hb J Meerut an alpha chain variant. In our study mean P2 values were 3.52 ± 1.05 while mean P3 values were 4.67 ± 1.43. No abnormal variant was detected in the P3 window. Four rare variants were picked up in this study these included Compound heterozygous delta beta/ beta thalassemia, HbD Iran, heterozygous HbO Indonesia , Hb G Sriraj /O Arab. HbD Iran The first rare variant picked up was Hb D Iran in a lady referred for antenatal screening. She had a Hb of 13.2gm/dl, TRBC-4.39 million/mm3 and normal RBC indices with MCV 89.1 fl, MCH 30 pg, MCHC 33.8gm/dl. On HPLC HbA2 was 41.1% with a retention time of 3.62 ,Hb A of 49.8%. On the HbCZE this abnormal haemoglobin was seen in the D window with HbA2 of 1.8%. Her sickling test was negative. Based on this she was diagnosed as a case of Hb D Iran. Reportedly in HPLC, nine abnormal Hbs elute in the Hb A2 window (3.27–3.83 as per the manufacturer's guidelines in the operating software): Hb Deer Lodge, Hb Lepore, Hb D Iran Hb E, Hb Hamadan, Hb Osu-Christiansborg, Hb Tianshu, Hb G Honolulu and Hb G Copenhagen. Among these, Hb Deer Lodge, Hb Lepore and Hb D Iran elute prior to the standard RT of Hb A2 (3.6 min) while others have higher RT to that of Hb A2. Interestingly, Hb Lepore has the lowest average quantity (7–15%) followed by Hb G Honolulu (about 15% of total hemoglobin quantity) and Hb E (about 30% of total hemoglobin in absence of α-thalassemias). All the other variants eluting in the Hb A2 window have variant hemoglobin quantities higher than 30% on average under heterozygous conditions, making it difficult to distinguish in HPLC. Amongst these, Hb D Iran has been reported to elute in this window at a RT of 3.49–3.58 min; almost in the middle of the window (3.27–3.83).A number of reasons contribute to the apparent low prevalence of Hb D Iran, more so because Hb D Iran when in heterozygous and homozygous form is clinically asymptomatic except for cases of compound heterozygosity with β/Hb D Iran where there is mild to moderate anemia and easy fatiguability. Majority of homozygous and heterozygous cases are detected retrospectively as a part of extended family screening. Hence most of the Hb D Iran cases are missed because of its asymptomatic phenotype.(7) 2. Hb G-Sriraj /O Agenogi The suspected Hb G-Sriraj /O Agenogi where the HPLC showed a peak in the C window of 87.7% with a retention time of 4.98 seconds an HbA2 of 7.7 % while on HbCZE HbA2 was 3.3% and a peak in the E window was found of 91.6%.( Figure B) On the HPLC O Arab, O Indonesia, Constant Spring, Agenogi, Sriraj may be found in the C window with a retention time ranging from 4.90 to 5.30. While on the HbCZE Hb E, Hb Köln (Ube-1), Hb Buenos Aires (minor peak), Hb Agenogi, Hb G-Sriraj, Hb Santa Ana, Hb A2-Babinga, Hb M-Saskatoon (minor peak), “M-Iwate” Hb A2 variant, denatured Hb are found.(8) He had a Hb of 7.5gm/dl, TRBC-4.01million/mm3, HCT 23, MCV of 57.6%, MCH 19pg, MCHC -32.5. bilirubin was raised at 1.40mg/dl but no splenomegaly was present. Based on this a provisional diagnosis of Hb G-Sriraj /O Arab was rendered. Due to rarity the exact incidence is not known. The carriers in the heterozygous state are clinically asymptomatic. Nonetheless, its complex interaction with other β-thalassaemia could give rise to different clinical phenotypes, ranging from mild thalassaemia intermedia to thalassaemia major.(7) The establishment of the diagnosis is challenging and laborious and the only confirmatory test is by molecular analysis. 3. Hb O Indonesia The third rare case a case of Hb O Indonesia had microcytic hypochromic indices with Hb of 9.7g/dl, MCV of 77.6, MCH of 24pg, MCHC of 31.1g/dl. This again was an ANC case referred for screening. The HBCZE showed HbA of 86.2% and HbS of 12% HbA2-2.3. The HPLC on the other hand showed an abnormal peak of 11.1% in the C window with a RT of 4.86 and HbA of 78.5 ,HbA2 of 2.3. Sicking test was negative. Hemoglobin O (HbO) is a rare type of hemoglobin in which there is a substitution of glutamic acid by lysine but at different positions. In Hb O Indonesia this replacement is at the position 116 of the beta globin chain.Hemoglobin O Indonesia is a was first reported in the Bugis population of Sulawesi Island in Indonesia, and hence designated originally as hemoglobin Buginese-X thereafter phenotypically similar mutations were found in Iran and Italy. HbO Indonesia is asymptomatic however in heterozygous states especially with Hb S there may be mild sickle cell trait.(7,8) 4. Compound heterozygous delta beta/ beta-thalassemia The compound heterozygous delta beta/ beta-thalassemia was picked up, on HbCZE and had values of 94.7% HbF, 1.6% HbA2 and acetylated Hb of 3.7% in the Z8 window. On HPLC there was HbF of 88.1%, HbA2 of 2.3%, and HbA of 5.5%. Two unknown peaks were found at retention times of 0.68 and 0.98 respectively with the percentage area of 0.2 and 0.4. Her Hb was 5.3% gm/dl ,TRBC was 2.33 million/mm3 and her indices were microcytic hypochromic with MCV of 77.7 fl, MCH of 23pg, and MCHC of 29.3g/dl. RDW was 26.5, based on the family screening studies she was diagnosed as compound heterozygous delta beta / beta-thalassemia with one parent showing raised HbF and the other parents being a beta thalassemia trait with raised HbA. Delta beta thalassemia results from the deletion of both delta and beta genes, homozygotes for δβ-thalassemia have 100% HbF and, because of the increased HbF have a thalassemia intermedia picture rather than thalassemia major. It is necessary to distinguish it from hereditary persistence of fetal hemoglobin (HPFH) which also has 100% HbF but is phenotypically different as HPFH is asymptomatic in contrast with delta beta thalassemia. The phenotype of delta beta thalassemia heterozygotes is similar to BTT but the HbA2 percentage is often normal. And HbF is raised varying from 5% to 20%.The incidence is unknown as only a handful of case reports are available. These rare variants and their corresponding findings on HPLC and HbCZE are depicted in table 3 below Table 3. Clinical and laboratory findings in the rare variants picked in this study. CLINICAL FINDINGS HPLC HbCZE DIAGNOSIS Antenatal screening Normocytic Normochromic indices HbA2- 41.1%(RT 3.62) Hb A - 49.8% D window -43.4% HbA2 of 1.8% HbA 54.8% Hb D Iran Microcytic hypochromic anemia , h/o gall stones C peak 87.7 (RT 4.98) HbA2- 7.7% HbA -3.1% E window-91.6% HbA2 -3.3% HbA- 3.2% HbG-Sriraj /O Agenogi Microcytic hypochromic anaemia HbF of 88.1% HbA2 of 2.3%, HbA of 5.5%. Hb F- 94.7% HbA2- 1.6% Acetylated Hb - 3.7% Compound heterozygous delta beta/beta-thalassemia Microcytic hypochromic anaemia abnormal peak of 11.1% (RT4.86) HbA-78.5 HbA2-2.3 HbA -86.2% HbS of 12% HbA2-2.3 Hb O Indonesia On comparing the HbA2 values between HbCZE and HPLC, HPLC showed higher values for HbA2 with a median value of 2.9% as compared to HbCZE where the value was 2.65%. The mean HbA2 for HbCZE was 3.21 ± 1.35%. In the case of cumulative HbA2 for HPLC, it was higher because the HbE, HbD Iran, and glycated HbS also fell in the HbA2 window thereby raising the value. When HbA2 values were compared by HbCZE in patients with HbS and without HbS the mean HbA2 was higher in patients with HbS being 3.44 ± 1.25% in patients with HbS while in patients without HbS it was 3.2 ± 1.35%. These were not statistically significant. When the same values were compared on the HPLC machine the mean for patients with HbS was 4.07 ± 1.22% while for those without HbS (also excluding HbE/HbC/HbD-Iran) it was3.38 ± 1.18% and this difference was statistically significant. We know that glycated adducts have a retention time similar to HbA2 thereby falsely elevating the percentage of HbA2 in the case of HbS on the HPLC machine and this was the reason for these elevated values. On correlating HbA2 between HPLC and HbCZE values in patients with HbA2 <3.5% the correlation coefficient was 0.867 with a significant p-value of 3.5 %the correlation coefficient was 0.369 with a p-value of <0.001 thereby implying the poor correlation between HbA2 values between HbCZE and HPLC at values over 3.5%.This is depicted in Figure 1. which compares HbA2 using HPLC and HbCZE with values of HbA2 3.5% As Hb E, HbD Iran also fell into the HbA2 window in the HPLC this may lead to elevated values as already discussed. The bias for low HbA2 3.5% it was - 8.0012. The HbF agreement within both the methods was good as evidenced by Figure 2. Figure 1. Comparison of HbA2 using HPLC and HbCZE with values of HbA2 3.5% Fig 2. Comparison of HbF using HPLC and HbCZE showing a good agreement with both methods. Discussion Haemoglobin fraction analysis cation-exchange HPLC has the advantage of quantifying Hb F and Hb A2 along with haemoglobin variant screening in a single, highly reproducible system.(13) It is simple automated, with internal sample preparation, superior resolution, and rapid assay time and accurate quantification. HPLC will accurately quantify Hb A2 in the presence of Hb C, but not in the presence of Hb E or glycated Hb S. The disadvantages are the higher capital cost, It cannot separate HbE, Hb Lepore, HbD Iran, Hb Korle blue as these elute in the HbA2 window. The glycosylated variants and acetylated variants have retention times different from the non-glycosylated and non-acetylated variants. Hb F may merge with peaks resulting from glycosylated HbA and therefore may not be detected if less than 0.6%. Glycosylated Hb A can falsely raise HbF. Similarly, glycosylated Hb S can elute in the HbA2 window falsely elevating it. (14) Bilirubin is the sample can lead to peaks in the same peak as HbH, Hb Barts and acetylated HbF.HbCZE is a relatively newer technique approved by FDA is 2007. It can measure HbA2 in the presence of HbE but not in the presence of HbC. HbCZE patterns can be read-only if HbA and HbA2 are present in the sample and therefore mixing studies have to be carried out if it is absent.(15) In the presence of HbS measured HbA2 is lower by HbCZE as compared to HPLC, while the reverse happens in the presence of HbD. Table 4 compares the salient features of our study comapred with those of similar studies conducted using HPLC and HbCZE. Table 4. Comparison of High performance liquid chromatography and Haemoglobin capillary zone electrophoresis in diagnosing thalassemias and haemoglobinopathies STUDY SALIENT FINDINGS Higgins, Khajuria & Mack(11) HbA2 is lower with HbCZE(variant II) as compared to HPLC(Capillarys 2) HbA2 in pts with HbS, heterozygous and homozygous similar by both HbCZE better quantifying HbA2 in HbE, HbD Punjab traits HbA2 upper reference range to be 3.1% by CE as compared to 3.6% by HPLC Hafiza Alauddin et al.(16) Normal ranges for HbA2 and HbF by HbCZE lower than HPLC HbA2 levels for HbE heterozygotes are higher than that of normal but lower than beta thal heterozygotes Mais et al (17) HbA2 of patients with the HbE trait was higher than normal HbA2 of HbE homozygotes was higher than heterozygotes HbE level was 24.28% by CE which was much lower than that of HPLC Van Delft et al (10) HbA2 higher( 2.67) with Variant II(2.51) as compared to Capillarys Elevated or overestimated HbA2 fractions are measured in the presence of HbS Keren D.F, et al(12) Hb A levels were similar on both HPLC and Sebia HBCZE HbA2levels were higher by CE than by HPLC In cases with HbS HbA2 levels were greater by HPLC than HbCZE HbA2 was occasionally not separated from HbC by CE but did separate from HbE by CE Greene DN et al (9) Hb A measurements agreed between HPLC (Variant II) and HbCZE(Sebia) Hb F measurement agreement was also good If no variant was present Hb A2concentrations showed excellent agreement Hb A2 bias was concentration-dependent: at low concentrations, a low bias was observed; at high concentrations, a high bias was observed Our Study Hb F, HbS measurement agreement was good between HPLC and HbCZE HbA2levels were higher by HbCZE than by HPLC In cases with HbS HbA2 levels were higher on HPLC than in those without HbA2, no significant variation on HbCZE Once variants are excluded the correlation is better at lower HbA2 values than at a higher value Conclusion Being an important cause of morbidity and mortality haemoglobinopathies impose a heavy burden on families and the health sector in our country. The identity of a haemoglobin variant is generally inferred from its electrophoretic mobility, its quantity, and the patient’s ethnic background however definite identification can be achieved only by DNA analysis or amino acid sequencing. Diagnostic algorithms should be developed keeping in mind the geographical locale, the kind of health setup primary secondary or tertiary and available resources.We have found HPLC and HbCZE to be complementary techniques and use both routinely. Normal ranges and means normal values differ between different methods and different manufacturers. This study his illustrates that each laboratory should have an alternative method adapted to each case and keep in mind the performance characteristics of each method.It appears that an extensive molecular work up of the β globin gene is the only definite method to detect borderline HbA2 β thalassemia carriers, more so in populations that have a high prevalence. Declarations Author contributions Concept : Somasundaram Venkatesan Data Curation : Gurpreet Kaur, Paresh Singhal Writing : Gurpreet Kaur, Ankur Ahuja, Kanwaljeet Singh Editing : Michael John K, Vikram Singh Funding : This research received no external funding Informed consent statement : Informed consent was obtained from the subject Conflicts of interest : NIL Human ethics : Certificate obtained from institutional ethics committee Data Availability declaration References Williams TN, Weatherall DJ. World distribution, population genetics and health burden of the hemoglobinopathies. Cold Spring Harb Prospects Med 2012;2. a011692 Bain B, Bates I, Laffan M, eds. Dacie and Lewis Practical Haematology. 12 th ed. UK: Elsevier; 2017 International Committee For Standardization In Haematology. Recommendations for selected methods for quantitative estimation of Hb A2 and for Hb A2 reference preparation. Br J Haematol 1978;38: 573–8. Colah R, Gorakshakar A, Nadkarni A. 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Interlaboratory comparison of current high-performance methods for HbA2. Int J Lab Hematol. 2012 Aug;34(4):362-8 Borbely N, Phelan L, Szydlo R, Bain B. Capillary zone electrophoresis for haemoglobinopathy diagnosis. J Clin Pathol. 2013 Jan;66(1):29-39. Hafiza A, et al. HbA2 levels in normal, beta-thalassaemia and haemoglobin E carriers by capillary electrophoresis. Malays J Pathol. 2012 Dec;34(2):161-4 Mais et al. The Range of Hemoglobin A(2) in Hemoglobin E Heterozygotes as Determined by Capillary Electrophoresis. American journal of clinical pathology.2009; 132. 34-8. 10. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4138194","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":289055662,"identity":"e52994a2-9476-446b-bec4-24cb741551e9","order_by":0,"name":"Gurpreet Kaur","email":"","orcid":"","institution":"Armed Forces Medical college","correspondingAuthor":false,"prefix":"","firstName":"Gurpreet","middleName":"","lastName":"Kaur","suffix":""},{"id":289055663,"identity":"99732b71-3470-4de9-b783-11a40d03493f","order_by":1,"name":"Ankur Ahuja","email":"","orcid":"","institution":"Armed Forces Medical college","correspondingAuthor":false,"prefix":"","firstName":"Ankur","middleName":"","lastName":"Ahuja","suffix":""},{"id":289055664,"identity":"22fd99aa-3ec1-44c2-9321-7f405a06ab10","order_by":2,"name":"Somasundaram Venkatesan","email":"","orcid":"","institution":"Armed Forces Medical college","correspondingAuthor":false,"prefix":"","firstName":"Somasundaram","middleName":"","lastName":"Venkatesan","suffix":""},{"id":289055665,"identity":"a98d0d99-5356-4057-baa6-bed6187ae83d","order_by":3,"name":"Paresh Singhal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYLACxgYQmcDA8AFIsbGTooVxBkgLMylamHlANCEt5uxnHz78ucMuX7c9+eBnm1/b5PmYGRg/fMzBrcWyJ93YmPdMsuW2M8+SpXP7bhu2MTMwS87chluLwYE0NmnGNmYDsxs5Zsy5PbeBbKB3ePFpOf+M/efPtnqIFsue2/aEtdxIY2PgbTsM0cLw43YiQS2WM54xS/O2HTcwA/pFsrfhdnIbM2MzXr+Y86cxfvzZVm1gdjz54Icff27bzm9vPvjhIz6HofAY28BkA271GFoY/uBVPApGwSgYBSMUAAAoy0+oMycOXwAAAABJRU5ErkJggg==","orcid":"","institution":"Armed Forces Medical college","correspondingAuthor":true,"prefix":"","firstName":"Paresh","middleName":"","lastName":"Singhal","suffix":""},{"id":289055666,"identity":"d49d0874-dcdf-4962-9acc-09334b902a5c","order_by":4,"name":"Kanwaljeet Singh","email":"","orcid":"","institution":"Armed Forces Medical college","correspondingAuthor":false,"prefix":"","firstName":"Kanwaljeet","middleName":"","lastName":"Singh","suffix":""},{"id":289055667,"identity":"e93a2668-343d-4fd6-849e-b3767f5cb402","order_by":5,"name":"Michael John Kanaimparampil","email":"","orcid":"","institution":"Armed Forces Medical college","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"John","lastName":"Kanaimparampil","suffix":""},{"id":289055668,"identity":"9dc8e363-a9d9-4bcd-82c6-930cd5edb958","order_by":6,"name":"Vikram Singh","email":"","orcid":"","institution":"Armed Forces Medical college","correspondingAuthor":false,"prefix":"","firstName":"Vikram","middleName":"","lastName":"Singh","suffix":""}],"badges":[],"createdAt":"2024-03-20 15:20:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4138194/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4138194/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54594595,"identity":"118c09e7-a392-43f3-abb8-dd832a5bb58f","added_by":"auto","created_at":"2024-04-12 18:28:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":30287,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of HbA2 using HPLC and HbCZE with values of HbA2 \u0026lt;3.5% and \u0026gt;3.5%\u003c/p\u003e","description":"","filename":"F1.png","url":"https://assets-eu.researchsquare.com/files/rs-4138194/v1/31a926e8981edf9a6f397e9b.png"},{"id":54594588,"identity":"55d72d61-2fe5-44ae-b13d-b541406fee87","added_by":"auto","created_at":"2024-04-12 18:28:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13027,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of HbF using HPLC and HbCZE showing a good agreement with both methods.\u003c/p\u003e","description":"","filename":"F2.png","url":"https://assets-eu.researchsquare.com/files/rs-4138194/v1/1d53803d5b2d0de7913448ba.png"},{"id":54595098,"identity":"ca81c77f-86f5-43cd-b520-d53ebef3d5e4","added_by":"auto","created_at":"2024-04-12 18:36:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":439546,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4138194/v1/0dde6301-c9d4-423e-b796-749ff666abd7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Diagnosis of Hemoglobinopathies and thalassemias using both Haemoglobin capillary zone electrophoresis and High performance liquid chromatography - An experience from a tertiary care hospital in Northern India","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSickle cell disease and thalassemia are the most common clinically significant haemoglobinopathies and the carriers of these disorders represent approximately 5.0% of the world\u0026rsquo;s population.The thalassemias and hemoglobinopathies are commonly encountered in areas where malaria is endemic because abnormal genes offer protection against malaria. They are classified as per the type of mutation (insertion-deletion, base change) or the affected globin chain (alpha chain, beta chain) structural haemoglobin variants including those involved in sickle cell disease, haemoglobin C (HbC) disease, and haemoglobin E (HbE) disease being the commonest Over 300,000 infants with severe Hb disorders are born each year(1-3).\u0026nbsp;Thalassemia and hemoglobinopathy are the major health concern in the Indian subcontinent as the prevalence rate of beta thalassemia mutations is as high as 17% in some populations. (4) Hb E-beta thalassemia and sickle-cell anemia are also common with high incidence in tribal populations.\u003c/p\u003e\n\u003cp\u003eThe detection and characterization of a haemoglobinopathy involves a 3 tier work up. (1) Full blood count (2)Special haematological tests (3)Molecular testing.The value of routine haematological analysis along with morphology and red cell indices is a must for the part of work up in any hemoglobinopathy. Reticulocyte counts, HbH preparations, Heinz body preparations, solubility test for HbS, unstable haemoglobin testing using \u0026nbsp; Isopropanol or heat stability test, Dichlorophenol Indophenol test (DCIP) for HbE screening, spectroscopic tests for unstable haemoglobins causing methemoglobinemia, P50 determination for oxygen (i.e. Oxygen affinity test), oxygen dissociation curves, etc are important tests to be performed in the testing for haemoglobinopathies.Most centres in India reporting the carrier frequencies of beta thalassemia and other haemoglobinopathies now use CBC and HPLC analysis of haemoglobins. Other methods include HbCZE, immunodiffusion (RID) and IFE (Isoelectric focusing).Molecular diagnosis of thalassemia is mainly applied for confirmation of screening results, for clarification of complicated cases, and or prenatal diagnosis. It is recommended that all positive screening results are confirmed through DNA analysis, and results should be interpreted altogether, including the evaluation of haematology and family history. Guidelines as per the ICSH for hemoglobinopathy screening not only recommend the use of quantitative techniques for thalassemia detection, they also stress the use of a confirmatory method when a Hb variant is detected. In an ideal setting, both techniques can be met by a combination of \u0026nbsp;HPLC and HbCZE.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003eFrom March 2019 to December 2020,\u0026nbsp;data from 536 whole blood samples sent to our institute for physician-ordered hemoglobinopathy screening were studied. The samples included patients being evaluated for microcytic hypochromic anaemia. Suspected iron deficiency anaemia unresponsive to iron therapy, prenatal/ antenatal screening, or sickle cell disease treatment and monitoring. High-Pressure Liquid Chromatography\u003c/p\u003e\n\u003cp\u003e(HPLC) was performed using Bio-Rad Variant II \u0026beta;-thalassemia Short Program (Bio-Rad, Hercules, CA), while Haemoglobin capillary zone electrophoresis (HBCZE) \u0026nbsp;was performed using Mini\u0026nbsp;Flex Piercing analyzer( Sebia trivitron).\u0026nbsp;Cases of human Immunodeficiency virus infection (HIV) on highly active human antiretroviral therapy (HAART), bone marrow failure syndromes (BMFS) Myelodysplastic syndromes (MDS) were excluded.A signed consent form was obtained from all the patients included in the study. Detailed clinical history and family history were obtained from each patient. Past history of blood transfusion, if present, was noted.Blood samples were collected in ethylene diamine tetrachloride acetate (EDTA) vials. For each patient, a peripheral blood smear (PBS) was prepared and stained with Leishman stain and observed \u0026nbsp;for morphology to supporting \u0026nbsp;the diagnosis of hemoglobinopathies. Hb variant analysis was performed \u0026nbsp;for various hemoglobinopathies and variants using HPLC and HBCZE. Confirmatory tests such as acid and sickling test, solubility test, and brilliant cresyl blue test for HbH inclusions were performed as and when required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh-Pressure Liquid Chromatography\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHPLC analysis was performed using the manufacturer\u0026apos;s instructions for the Bio-Rad Variant II \u0026beta;-thalassemia Short Program (Bio-Rad, Hercules, CA), which separates haemoglobin variants by cation exchange chromatography using a salt gradient. The VARIANT II is a fully automated HPLC system that can be used to separate and determine area percentages for haemoglobins A2 and F and to provide qualitative determinations of abnormal haemoglobins. The identification of these haemoglobin variants is made using retention time windows such as a \u0026quot;D-Window,\u0026quot; \u0026quot;S-Window,\u0026quot; and \u0026quot;C-Window.\u0026quot; \u0026nbsp;In the VARIANT II \u0026beta;-thalassemia Short Program the samples are automatically mixed and diluted on the VARIANT II Sampling Station (VSS) and injected into the analytical cartridge. Different haemoglobins vary in their retention times i.e. the time from application to elution from the column, their characteristic. Each sample run takes about six and half minutes and nine samples may be run in one hour. (5,7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHaemoglobin capillary zone electrophoresis \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaemoglobin electrophoresis is in many ways an intermediary type of technique between classical zone electrophoresis and liquid chromatography which separates analytes according to the pH of the electrolyte solution and electroosmotic flow. For this study the capillary Mini Flex Piercing analyzer, Sebia trivitron was used. This instrument uses the principle of capillary electrophoresis in a free solution. The instrument is equipped to resuspend, lyse, separate, and analyze whole blood samples for variants collected in EDTA tubes. The haemoglobin bands were detected by absorption photometry and optical density measurements and converted to a migration image. This is then displayed as an electropherogram. The migration position for each haemoglobin in each sample is standardized relative to the position of the haemoglobin A and A2 bands and is measured in arbitrary units between 0 and 300. \u0026nbsp;The distinct peaks in the migration image are assigned to one of 15 zones and are quantified as percentage haemoglobins A, F, A2 and C are provisionally identified and colour coded. 28 samples can be processed in one go and \u0026nbsp;eight samples are run per hour. (6,7)\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 536 patients with suspected haemoglobinopathy were studied. The age group of the study population ranged from 0.2 to 73 years with a mean age of 26.43 years. There were more females in the study group 377 females (70.34%) and 159 males (29.66%) owing to a large number of antenatal cases that were screened. The most common subset was patients referred for antenatal checkups 224(41.79%). Clinically pallor had been documented in 84 (15.67%) patients and 112(20.90%) cases had been sent as family screening cases.17 cases out of 536 (3.17%) presented had jaundice on presentation while a history of transfusion was documented in 53 cases. Out of a total of 536 patients that were screened 141 patients were detected to have underlying thalassemia or haemoglobinopathy. The majority of these patients were BTT 97(68.79%) followed by compound heterozygous Hb E -beta-thalassemia 15(10.64%) and heterozygous E state 12(8.51%). There were 02 (1.42%) homozygous beta-thalassemia patients and 03 (2.13%) each with Homozygous HbS and Heterozygous sickle cell state. One each (0.71%) of compound heterozygous HbS beta-thalassemia, compound heterozygous delta beta-thalassemia, HbD \u0026nbsp;Iran, Heterozygous HbO-Indonesia, Haemoglobin G Sriraj /O Arab were also identified. Three samples had a previous history of transfusion and 01 sample showed a carryover of HbD of 2.7%. A repeat HPLC/HbCZE was advised for these patients but they were lost to follow up. The distribution of subgroups is depicted in table 1. The erythrocyte indices of the commonest subgroups are shown in table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 01:-Distribution of subgroups of study subjects\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"64.64646464646465%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubgroups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.171717171717173%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFrequency\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"64.64646464646465%\"\u003e\n \u003cp\u003eHomozygous beta thalassemia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.171717171717173%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e1.42%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"64.64646464646465%\"\u003e\n \u003cp\u003eHeterozygous beta thalassemia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.171717171717173%\"\u003e\n \u003cp\u003e97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e68.79%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"64.64646464646465%\"\u003e\n \u003cp\u003eHeterozygous E state\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.171717171717173%\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e8.51%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"64.64646464646465%\"\u003e\n \u003cp\u003eCompound heterozygous Hb E -beta-thalassemia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.171717171717173%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e10.64%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"64.64646464646465%\"\u003e\n \u003cp\u003eHomozygous HbS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.171717171717173%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e2.13%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"64.64646464646465%\"\u003e\n \u003cp\u003eHeterozygous sickle cell state\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.171717171717173%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e2.13%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"64.64646464646465%\"\u003e\n \u003cp\u003eCompound heterozygous HbS beta-thalassemia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.171717171717173%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e0.71%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"64.64646464646465%\"\u003e\n \u003cp\u003eCompound heterozygous delta beta /beta-thalassemia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.171717171717173%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e0.71%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"64.64646464646465%\"\u003e\n \u003cp\u003eHbD \u0026nbsp; Iran\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.171717171717173%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e0.71%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"64.64646464646465%\"\u003e\n \u003cp\u003eHeterozygous HbO-Indonesia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.171717171717173%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e0.71%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"64.64646464646465%\"\u003e\n \u003cp\u003eHaemoglobin G Sriraj / O Arab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.171717171717173%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e0.71%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"64.64646464646465%\"\u003e\n \u003cp\u003ePost transfusion sample /carry over samples\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.171717171717173%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e2.84%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"64.64646464646465%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.171717171717173%\"\u003e\n \u003cp\u003e141\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.181818181818183%\"\u003e\n \u003cp\u003e100.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Erythrocyte indices of the commonest subgroups\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eheterozygous beta-thalassemia, Compound heterozygous Hb E beta-thalassemia, Heterozygous E state\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"90%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.89795918367347%\"\u003e\n \u003cp\u003e\u003cstrong\u003eErythrocyte indices\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHeterozygous beta thalassemia(n=97)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.408163265306122%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompound heterozygous Hb E beta-thalassemia(n=15)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHeterozygous E state(n=12)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.89795918367347%\"\u003e\n \u003cp\u003eHaemoglobin(g/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\"\u003e\n \u003cp\u003e11.3 \u0026plusmn; 2.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.408163265306122%\"\u003e\n \u003cp\u003e8.13 \u0026plusmn; 3.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\"\u003e\n \u003cp\u003e11.61 \u0026plusmn; 1.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.89795918367347%\"\u003e\n \u003cp\u003eTRBC(x10\u003csup\u003e12\u003c/sup\u003e/l )\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\"\u003e\n \u003cp\u003e5.57 \u0026plusmn; 1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.408163265306122%\"\u003e\n \u003cp\u003e4.52 \u0026plusmn; 1.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\"\u003e\n \u003cp\u003e4.62 \u0026plusmn; 1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.89795918367347%\"\u003e\n \u003cp\u003eHCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\"\u003e\n \u003cp\u003e36.62 \u0026plusmn; 6.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.408163265306122%\"\u003e\n \u003cp\u003e29.11 \u0026plusmn; 11.02.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\"\u003e\n \u003cp\u003e35.93 \u0026plusmn; 4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.89795918367347%\"\u003e\n \u003cp\u003eMean corpuscular haemoglobin (fl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\"\u003e\n \u003cp\u003e55.3 \u0026plusmn;85.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.408163265306122%\"\u003e\n \u003cp\u003e61.32 \u0026plusmn;8.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\"\u003e\n \u003cp\u003e73.79 \u0026plusmn;4.38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.89795918367347%\"\u003e\n \u003cp\u003eMean corpuscular haemoglobin \u0026nbsp;(pg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\"\u003e\n \u003cp\u003e20.67 \u0026plusmn;3.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.408163265306122%\"\u003e\n \u003cp\u003e17.83 \u0026plusmn; 3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\"\u003e\n \u003cp\u003e23.81 \u0026plusmn; 1.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.89795918367347%\"\u003e\n \u003cp\u003eMean corpuscular haemoglobin concentration (g/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\"\u003e\n \u003cp\u003e32.67 \u0026plusmn; 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.408163265306122%\"\u003e\n \u003cp\u003e28.79 \u0026plusmn; 2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.346938775510203%\"\u003e\n \u003cp\u003e31.48 \u0026plusmn; 2.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eOn HbCZE the haemoglobin A values in the study group ranged from 3.2-99% with a mean HbA of 93.74 \u0026plusmn; 1.Haemoglobin A2 (HbA2) constitutes less than 3% of the total haemoglobin (Hb) in adults and has almost no physiological importance however the determination of Hb A2 levels is important to diagnose the beta thalassemia trait (BTT).The haemoglobin A2 values in the study group ranged from 3.2-99% with a mean HbA of 93.74 \u0026plusmn; 12.66 %. For the sake of statistical analysis HbA2 values were divided into the following groups for ease of comparison \u0026lt;2%, 2.0-3.3%, 3.4-3.7%, 3.8-7.0% and \u0026gt;7% with HbA2 \u0026lt; 2% indicative of IDA, alpha thalassemia, HbH disease \u0026delta;\u0026beta; thalassaemia (if haemoglobin F was also elevated), 2.0 - 3.3% was essentially the normal values for HbA2, HbA2 of 3.4-3.7% was indicative of a borderline elevated HbA2, or severe iron deficiency in \u0026beta; thalassaemia trait. \u0026beta; thalassaemia trait with additional \u0026delta; chain variant may also have lower values of HbA2. In the presence of haemoglobin S, HbA2 values are raised on HPLC and may fall in this borderline zone. Haemoglobin A2 values of \u0026gt;7.0% are rare and a structural variant needs exclusion after repeating HbA2 estimation. The majority of the patients 340 (63.43%) had levels of HbA2 within the normal range i.e. 2.0-3.3 % while the second most commonest was HbA2 levels of 3.8-7.0% totalling to 149 pts (27.80%). Six pts had borderline values of between 3.4 \u0026nbsp;and 3.7% only two pts (0.37%) had values over 7%.\u003c/p\u003e\n\u003cp\u003eOn HPLC 352 (65.67%) pts had normal HbA2 values, and 136(25.37%) pts had HbA2 which fell into the range of 3.8-7.0%. 31 pts had values that were more than 7% and these were later sub-grouped based on the percentage of HbA2 present as Hb Lepore, HbE, and HbD Iran coeluate in this window. Values of HbA2 between 10-14% are indicated of Hb Lepore, 25-40% Hb E heterozygous, 44-48% HbD Iran and 70-90 % E homozygous. While the P2 window has the glycated Hb eluting in it, in the P3 window values up to 6% are acceptable 6-12% are indicative of sample deterioration and 15-25% are indicative of Hb J Meerut an alpha chain variant. In our study mean P2 values were 3.52 \u0026plusmn; 1.05 while mean P3 values were 4.67 \u0026plusmn; 1.43. No abnormal variant was detected in the P3 window.\u003c/p\u003e\n\u003cp\u003eFour rare variants were picked up in this study these included Compound heterozygous delta beta/ beta \u0026nbsp;thalassemia, HbD Iran, heterozygous HbO Indonesia , Hb G Sriraj /O Arab.\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003e\u003cstrong\u003eHbD Iran\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe first rare variant picked up was Hb D Iran in a lady referred for antenatal screening. She had a Hb of 13.2gm/dl, TRBC-4.39 million/mm3 and normal RBC \u0026nbsp;indices with MCV 89.1 fl, MCH 30 pg, MCHC 33.8gm/dl. On HPLC HbA2 was 41.1% with a retention time of 3.62 ,Hb A of 49.8%. On the HbCZE this abnormal haemoglobin was seen in the D window with HbA2 of 1.8%. Her sickling test was negative. \u0026nbsp; Based on this she was diagnosed as a case of Hb D Iran. Reportedly in \u0026nbsp;HPLC, nine abnormal Hbs elute in the Hb A2 window (3.27\u0026ndash;3.83 as per the manufacturer\u0026apos;s guidelines in the operating software): Hb Deer Lodge, Hb Lepore, Hb D Iran \u0026nbsp;Hb E, Hb Hamadan, Hb Osu-Christiansborg, Hb Tianshu, Hb G Honolulu and Hb G Copenhagen. Among these, Hb Deer Lodge, Hb Lepore and Hb D Iran elute prior to the standard RT of Hb A2 (3.6 min) while others have higher RT to that of Hb A2. Interestingly, Hb Lepore has the lowest average quantity (7\u0026ndash;15%) followed by Hb G Honolulu (about 15% of total hemoglobin quantity) and Hb E (about 30% of total hemoglobin in absence of \u0026alpha;-thalassemias). All the other variants eluting in the Hb A2 window have variant hemoglobin quantities higher than 30% on average under heterozygous conditions, making it difficult to distinguish in HPLC. Amongst these, Hb D \u0026nbsp;Iran \u0026nbsp;has been reported to elute in this window at a RT of 3.49\u0026ndash;3.58 min; almost in the middle of the window (3.27\u0026ndash;3.83).A number of reasons contribute to the apparent low prevalence of Hb D Iran, more so because \u0026nbsp;Hb D Iran when \u0026nbsp;in heterozygous and homozygous form is clinically asymptomatic except for cases of compound heterozygosity with \u0026nbsp;\u0026beta;/Hb D Iran where there is \u0026nbsp;mild to moderate anemia and easy fatiguability. Majority of homozygous and heterozygous cases are detected retrospectively as a part of extended family screening. Hence most of the Hb D Iran cases are missed because of its asymptomatic phenotype.(7)\u003c/p\u003e\n\u003cp\u003e2.\u003cstrong\u003eHb G-Sriraj /O Agenogi\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe suspected \u0026nbsp;Hb G-Sriraj /O Agenogi where the HPLC showed a peak in the C window of 87.7% with a retention time of 4.98 seconds an HbA2 of 7.7 % while on HbCZE HbA2 was 3.3% and a peak in the E window was found of 91.6%.( Figure B) On the HPLC O Arab, O Indonesia, Constant Spring, Agenogi, Sriraj may be found in the C window with a retention time ranging from 4.90 to 5.30. While on the HbCZE Hb E, Hb K\u0026ouml;ln (Ube-1), Hb Buenos Aires (minor peak), Hb Agenogi, Hb G-Sriraj, Hb Santa Ana, Hb A2-Babinga, Hb M-Saskatoon (minor peak), \u0026ldquo;M-Iwate\u0026rdquo; Hb A2 variant, denatured Hb are found.(8) He had a Hb of 7.5gm/dl, TRBC-4.01million/mm3, HCT 23, MCV of 57.6%, MCH 19pg, MCHC -32.5. bilirubin was raised at 1.40mg/dl \u0026nbsp; \u0026nbsp;but no splenomegaly was present. Based on this a provisional diagnosis of Hb G-Sriraj /O Arab was rendered. \u0026nbsp;Due to rarity the exact \u0026nbsp;incidence is not known. The carriers in the heterozygous state are clinically asymptomatic. Nonetheless, its complex interaction with other \u0026beta;-thalassaemia could give rise to different clinical phenotypes, ranging from mild thalassaemia intermedia to thalassaemia major.(7) The establishment of the diagnosis is challenging and laborious and the only confirmatory test is by molecular analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.\u003cstrong\u003eHb O Indonesia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe third rare case a case of Hb O Indonesia \u0026nbsp; had microcytic hypochromic indices with Hb of 9.7g/dl, MCV of 77.6, MCH of 24pg, MCHC of 31.1g/dl. This again was an ANC case referred for screening. The HBCZE showed HbA of 86.2% and HbS of 12% HbA2-2.3. The HPLC on the other hand showed an abnormal peak \u0026nbsp;of 11.1% in the C window with a RT of \u0026nbsp;4.86 and HbA of 78.5 ,HbA2 of 2.3. Sicking test was negative. Hemoglobin O (HbO) is a rare type of hemoglobin in which there is a substitution of glutamic acid by lysine but at different positions. In Hb O Indonesia this replacement is at the position 116 of the beta globin chain.Hemoglobin O Indonesia is a was first reported in the Bugis population of Sulawesi Island in Indonesia, and hence designated originally as hemoglobin Buginese-X thereafter phenotypically similar mutations were found in Iran and Italy. HbO Indonesia is asymptomatic however in heterozygous states especially with Hb S there may be mild sickle \u0026nbsp;cell trait.(7,8)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Compound heterozygous delta beta/ beta-thalassemia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe compound heterozygous delta beta/ beta-thalassemia was picked up, on HbCZE and \u0026nbsp;had values of 94.7% HbF, 1.6% HbA2 and acetylated Hb of 3.7% in the Z8 window. On HPLC there was HbF of 88.1%, HbA2 of 2.3%, and HbA of 5.5%. Two unknown peaks were found at retention times of 0.68 and 0.98 respectively with the percentage area of 0.2 and 0.4. Her Hb was 5.3% gm/dl ,TRBC was 2.33 million/mm3 and her indices were microcytic hypochromic with MCV of 77.7 fl, MCH of 23pg, and MCHC of 29.3g/dl. RDW was 26.5, based on the family screening studies she was diagnosed as compound heterozygous delta beta / beta-thalassemia with one parent showing raised HbF and the other parents being a beta thalassemia trait with raised HbA. Delta beta thalassemia results from the deletion of both delta and beta genes, homozygotes for \u0026delta;\u0026beta;-thalassemia have 100% HbF and, because of the increased HbF \u0026nbsp;have a thalassemia intermedia picture \u0026nbsp;rather than thalassemia major. It is necessary to distinguish it from hereditary persistence of fetal hemoglobin (HPFH) which also has 100% HbF but is phenotypically different as HPFH is asymptomatic in contrast with delta beta thalassemia. The phenotype of delta beta thalassemia heterozygotes is similar to BTT but the HbA2 percentage is \u0026nbsp;often normal. And HbF \u0026nbsp;is raised varying from 5% to 20%.The incidence is unknown as only a handful of case reports are available.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese rare variants and their corresponding findings on HPLC and HbCZE are depicted in table 3 below\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Clinical and laboratory findings in the rare variants picked in this study.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"637\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.43171114599686%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; CLINICAL FINDINGS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.47252747252747%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHPLC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.1585557299843%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHbCZE\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.937205651491364%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDIAGNOSIS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.43171114599686%\" valign=\"top\"\u003e\n \u003cp\u003eAntenatal screening\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNormocytic Normochromic indices\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.47252747252747%\" valign=\"top\"\u003e\n \u003cp\u003eHbA2- 41.1%(RT 3.62)\u003c/p\u003e\n \u003cp\u003eHb A - 49.8%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.1585557299843%\" valign=\"top\"\u003e\n \u003cp\u003eD window -43.4%\u003c/p\u003e\n \u003cp\u003eHbA2 of 1.8%\u003c/p\u003e\n \u003cp\u003eHbA 54.8%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.937205651491364%\" valign=\"top\"\u003e\n \u003cp\u003eHb D Iran\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.43171114599686%\" valign=\"top\"\u003e\n \u003cp\u003eMicrocytic hypochromic anemia ,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eh/o gall stones\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.47252747252747%\" valign=\"top\"\u003e\n \u003cp\u003eC peak \u0026nbsp;87.7 (RT 4.98)\u003c/p\u003e\n \u003cp\u003eHbA2- 7.7%\u003c/p\u003e\n \u003cp\u003eHbA -3.1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.1585557299843%\" valign=\"top\"\u003e\n \u003cp\u003eE window-91.6%\u003c/p\u003e\n \u003cp\u003eHbA2 -3.3%\u003c/p\u003e\n \u003cp\u003eHbA- 3.2%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.937205651491364%\" valign=\"top\"\u003e\n \u003cp\u003eHbG-Sriraj /O Agenogi\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.43171114599686%\" valign=\"top\"\u003e\n \u003cp\u003eMicrocytic hypochromic anaemia\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.47252747252747%\" valign=\"top\"\u003e\n \u003cp\u003eHbF of 88.1%\u003c/p\u003e\n \u003cp\u003eHbA2 of 2.3%,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eHbA of 5.5%.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.1585557299843%\" valign=\"top\"\u003e\n \u003cp\u003eHb F- 94.7%\u003c/p\u003e\n \u003cp\u003eHbA2- 1.6%\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eAcetylated Hb - 3.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.937205651491364%\" valign=\"top\"\u003e\n \u003cp\u003eCompound heterozygous delta beta/beta-thalassemia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.43171114599686%\" valign=\"top\"\u003e\n \u003cp\u003eMicrocytic hypochromic anaemia\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.47252747252747%\" valign=\"top\"\u003e\n \u003cp\u003eabnormal peak of 11.1% (RT4.86)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eHbA-78.5\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;HbA2-2.3\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.1585557299843%\"\u003e\n \u003cp\u003eHbA -86.2%\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eHbS of 12%\u003c/p\u003e\n \u003cp\u003eHbA2-2.3\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.937205651491364%\" valign=\"top\"\u003e\n \u003cp\u003eHb O Indonesia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eOn comparing the HbA2 values between HbCZE and HPLC, HPLC showed higher values for HbA2 with a median value of 2.9% as compared to HbCZE where the value was 2.65%. The mean HbA2 for HbCZE \u0026nbsp;was 3.21 \u0026plusmn; 1.35%. In the case of cumulative HbA2 for HPLC, it was higher because the HbE, HbD Iran, \u0026nbsp;and glycated HbS also fell in the HbA2 window thereby raising the value. When HbA2 values were compared by HbCZE in patients with HbS and without HbS the mean HbA2 was higher in patients with HbS being 3.44 \u0026plusmn; 1.25% in patients with HbS while in patients without HbS it was 3.2 \u0026plusmn; 1.35%. These were not statistically significant. When the same values were compared on the HPLC machine the mean for patients with HbS was 4.07 \u0026plusmn; 1.22% while for those without HbS (also excluding HbE/HbC/HbD-Iran) it was3.38 \u0026plusmn; 1.18% and this difference was statistically significant. We know that glycated adducts have a retention time similar to HbA2 thereby falsely elevating the percentage of HbA2 in the case of HbS on the HPLC machine and this was the reason for these elevated values. On correlating HbA2 between HPLC and HbCZE values in patients with HbA2 \u0026lt;3.5% the correlation coefficient was 0.867 with a significant p-value of \u0026lt; 0.001 while in patients with HbA2 \u0026gt; 3.5 %the correlation coefficient was 0.369 with a p-value of \u0026lt;0.001 thereby implying the poor correlation between HbA2 values between HbCZE and HPLC at values over 3.5%.This is depicted in \u0026nbsp; Figure 1. which compares \u0026nbsp; HbA2 \u0026nbsp;using HPLC and HbCZE with values of HbA2 \u0026lt;3.5% and \u0026gt;3.5%\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;As Hb E, HbD Iran also fell into the HbA2 window in the HPLC this may lead to elevated values as already discussed. The bias for low HbA2 \u0026lt; 3.5% was -0.2882 while for higher values \u0026gt;3.5% it was - 8.0012. \u0026nbsp; The HbF agreement within both the methods was good as evidenced by Figure 2.\u003c/p\u003e\n\u003cp\u003eFigure 1. Comparison of HbA2 \u0026nbsp;using HPLC and HbCZE with values of HbA2 \u0026lt;3.5% and \u0026gt;3.5%\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFig 2. Comparison of HbF using HPLC and HbCZE showing a good agreement with both methods.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHaemoglobin fraction analysis cation-exchange HPLC has the advantage of quantifying Hb F and Hb A2 \u0026nbsp; along with haemoglobin variant screening in a single, highly reproducible system.(13) It is simple automated, with internal sample preparation, superior resolution, and rapid assay time and accurate quantification. HPLC will accurately quantify Hb A2 in the presence of Hb C, but not in the presence of Hb E or glycated Hb S. The disadvantages are the higher capital cost, It cannot separate HbE, Hb Lepore, HbD Iran, Hb Korle blue as these elute in the HbA2 window. The glycosylated variants and acetylated variants have retention times different from the non-glycosylated and non-acetylated variants. Hb F may merge with peaks resulting from glycosylated HbA and therefore may not be detected if less than 0.6%. Glycosylated Hb A can falsely raise HbF. Similarly, glycosylated Hb S can elute in the HbA2 window falsely elevating it. (14) Bilirubin is the sample can lead to peaks in the same peak as HbH, Hb Barts and acetylated HbF.HbCZE is a relatively newer technique approved by FDA is 2007. It can measure HbA2 in the presence of HbE but not in the presence of \u0026nbsp;HbC. HbCZE patterns can be read-only if HbA and HbA2 are present in the sample and therefore mixing studies have to be carried out if it is absent.(15) In the presence of HbS measured HbA2 is lower by HbCZE as compared to HPLC, while the reverse happens in the presence of HbD. Table 4 compares \u0026nbsp;the salient features of our study comapred with those of similar studies conducted using HPLC and HbCZE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4. Comparison of High performance liquid chromatography and Haemoglobin capillary zone electrophoresis in diagnosing thalassemias and haemoglobinopathies\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"106%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.242424242424242%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSTUDY\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"75.75757575757575%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSALIENT FINDINGS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.242424242424242%\" valign=\"top\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHiggins, Khajuria \u0026amp; Mack(11)\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"75.75757575757575%\" valign=\"top\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHbA2 is lower with HbCZE(variant II) as compared to HPLC(Capillarys 2)\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHbA2 in pts with HbS, heterozygous and homozygous \u0026nbsp;similar by both\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHbCZE better quantifying HbA2 in \u0026nbsp;HbE, HbD Punjab traits\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHbA2 upper \u0026nbsp;reference range to be 3.1% by CE as compared to 3.6% by HPLC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.242424242424242%\" valign=\"top\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHafiza Alauddin et al.(16)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"75.75757575757575%\" valign=\"top\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eNormal ranges for HbA2 and HbF by HbCZE lower than HPLC\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHbA2 levels for HbE heterozygotes are higher than that of normal but lower than beta thal heterozygotes\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.242424242424242%\" valign=\"top\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eMais et al (17)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"75.75757575757575%\" valign=\"top\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHbA2 of patients with the HbE trait was higher than normal\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHbA2 of HbE homozygotes was higher than heterozygotes \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHbE level was 24.28% by CE which was much lower than that of HPLC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.242424242424242%\" valign=\"top\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eVan Delft et al (10)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"75.75757575757575%\" valign=\"top\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHbA2 higher( 2.67) with Variant II(2.51) as compared to Capillarys\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eElevated or overestimated HbA2 fractions are measured in the presence of HbS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.242424242424242%\" valign=\"top\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eKeren D.F, et al(12)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"75.75757575757575%\" valign=\"top\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHb A levels were similar on both HPLC and Sebia HBCZE\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHbA2levels were higher by CE than by HPLC\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eIn cases with HbS HbA2 levels were greater by HPLC \u0026nbsp;than HbCZE\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHbA2 was occasionally not separated from HbC by CE but did separate from HbE by CE\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.242424242424242%\" valign=\"top\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eGreene DN et al (9)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"75.75757575757575%\" valign=\"top\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHb A measurements agreed between HPLC (Variant II) and HbCZE(Sebia) Hb F measurement agreement \u0026nbsp;was also good\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eIf no variant was present \u0026nbsp;Hb A2concentrations showed excellent agreement\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHb A2 bias was concentration-dependent: at low concentrations, a low bias was observed; at high concentrations, a high bias was observed\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.242424242424242%\" valign=\"top\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eOur Study\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"75.75757575757575%\" valign=\"top\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHb F, HbS measurement agreement \u0026nbsp;was \u0026nbsp;good between HPLC and HbCZE\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eHbA2levels were higher by HbCZE than by HPLC\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eIn cases with HbS HbA2 levels were higher on HPLC than in those without HbA2, \u0026nbsp;no significant variation on HbCZE\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eOnce variants are excluded the correlation is better at lower HbA2 values than at a higher value\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBeing an important cause of morbidity and mortality haemoglobinopathies impose a heavy burden on families and the health sector in our country. The identity of a haemoglobin variant is generally inferred from its electrophoretic mobility, its quantity, and the patient\u0026rsquo;s ethnic background however definite identification can be achieved only by DNA analysis or amino acid sequencing. Diagnostic algorithms should be developed keeping in mind the geographical locale, the kind of health setup primary secondary or tertiary and available resources.We have found HPLC and HbCZE to be complementary techniques and use both routinely. Normal \u0026nbsp; ranges and means normal values differ between different methods and different manufacturers. This study his illustrates that each laboratory should have an alternative method adapted to each case and keep in mind the performance characteristics of each method.It appears that an extensive molecular work up of the \u0026beta; globin gene is the only definite method to detect borderline HbA2 \u0026beta; thalassemia carriers, more so in populations that have a high prevalence.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eConcept : Somasundaram Venkatesan \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData Curation : Gurpreet Kaur, Paresh Singhal \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWriting : Gurpreet Kaur, Ankur Ahuja, Kanwaljeet Singh\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEditing : Michael John K, Vikram Singh\u003c/p\u003e\n\u003cp\u003eFunding : \u0026nbsp;This research received no external funding\u003c/p\u003e\n\u003cp\u003eInformed consent statement : Informed consent was obtained from the subject\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConflicts of interest : NIL\u003c/p\u003e\n\u003cp\u003eHuman ethics \u0026nbsp;: Certificate obtained from institutional ethics committee\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData Availability declaration\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWilliams TN, Weatherall DJ. World distribution, population genetics and health burden of the hemoglobinopathies. Cold Spring Harb Prospects Med 2012;2. a011692\u003c/li\u003e\n \u003cli\u003eBain B, Bates I, Laffan M, eds. Dacie and Lewis Practical Haematology. 12 th ed. UK: Elsevier; 2017\u003c/li\u003e\n \u003cli\u003eInternational Committee For Standardization In Haematology. Recommendations for selected methods for quantitative estimation of Hb A2 and for Hb A2 reference preparation. Br J Haematol 1978;38: 573\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eColah R, Gorakshakar A, Nadkarni A. Global burden, distribution and prevention of \u0026beta;-thalassemias and hemoglobin E disorders. Expert Rev Hematol. 2010;3:103\u0026ndash;17.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eVARIANT II b thalassemia short program instruction manual.\u003c/li\u003e\n \u003cli\u003eSebia (2013). HbA1c using the capillarys 2 flex piercing instrument. France, Sebia\u003c/li\u003e\n \u003cli\u003eBain BJ, Wild BJ, Stephens AD, et al. Variant haemoglobins: a guide to identification. 1st ed. West Sussex, UK: Wiley Blackwell; 2010.\u003c/li\u003e\n \u003cli\u003eJoutovsky A, Hadzi-Nesic J, Nardi MA. HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: a study of 60000 samples in a clinical diagnostic laboratory. \u003cem\u003eClin Chem.\u0026nbsp;\u003c/em\u003e2004;50:1736-1747\u003c/li\u003e\n \u003cli\u003eGreene DN et al \u0026nbsp;Comparison of Sebia Capillarys Flex capillary electrophoresis with the BioRad Variant II high pressure liquid chromatography in the evaluation of hemoglobinopathies. Clin Chim Acta. 2012 Aug 16;413(15-16):1232-8. doi: 10.1016/j.cca.2012.03.027. Epub 2012 Apr 10. PMID: 22515960\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eVan Delft P., Lenters E., Bakker-Verweij M., de Korte M., Baylan U., Harteveld C.L. \u0026amp; Giordano P.C. (2009) Evaluating five dedicated automatic devices for haemoglobinopathydiagnostics in multi-ethnic populations. International Journal of Laboratory Hematology31, 484\u0026ndash;495\u003c/li\u003e\n \u003cli\u003eHiggins T.N., Khajuria A. \u0026amp; Mack M. (2009) Quantification of HbA2 in patients with and without b-thalassemia and in the presence of \u0026nbsp;HbS, HbC, HbE and HbD Punjab haemoglobin variants. Comparison of two systems. American Journal of Clinical Pathology 131, 357\u0026ndash;362.\u003c/li\u003e\n \u003cli\u003eKeren D.F, et al. Comparison of Sebia Capillarys capillary electrophoresis with the Primus High-Pressure Liquid Chromatography in the evaluation of hemoglobinopathies. American Journal of Clinical Pathology 130,824\u0026ndash;831.\u003c/li\u003e\n \u003cli\u003eGalanello R and Cao A(1998) Relationship between genotype and phenotype: thalassemia intermedia.Ann NY Acad Sci 850, 325\u0026ndash;333\u003c/li\u003e\n \u003cli\u003ePaleari R, Gulbis B, Cotton F, Mosca A. Interlaboratory comparison of current high-performance methods for HbA2. Int J Lab Hematol. 2012 Aug;34(4):362-8\u003c/li\u003e\n \u003cli\u003eBorbely N, Phelan L, Szydlo R, Bain B. Capillary zone electrophoresis for haemoglobinopathy diagnosis. J Clin Pathol. 2013 Jan;66(1):29-39.\u003c/li\u003e\n \u003cli\u003eHafiza A, et al. HbA2 levels in normal, beta-thalassaemia and haemoglobin E carriers by capillary electrophoresis. Malays J Pathol. 2012 Dec;34(2):161-4\u003c/li\u003e\n \u003cli\u003eMais et al. The Range of Hemoglobin A(2) in Hemoglobin E Heterozygotes as Determined by Capillary Electrophoresis. American journal of clinical pathology.2009; 132. 34-8. 10.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Haemoglobinopathies, Haemoglobin capillary zone electrophoresis, high-performance liquid chromatography","lastPublishedDoi":"10.21203/rs.3.rs-4138194/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4138194/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Being an important cause of morbidity and mortality, thalassemias and hemoglobinopathies impose a heavy burden on families and the health sector in our country. It has has been estimated that in India, 0.37 per 1,000 fetuses have Hb disorder and more than 10,000 children are born annually with severe hemoglobinopathy and only around 10.0% are managed optimally, therefore screening and diagnosis of carriers is of paramount importance especially in an antenatal OPD.\nWhile Haemoglobin capillary zone electrophoresis (HbCZE) and High performance liquid chromatography (HPLC) are commonly used methods for diagnosis of these entities, there are far and few studies which attempt to compare these two techniques. Haemoglobin A2 (HbA2) constitutes less than 3% of the total hemoglobin (Hb) in adults and the determination of Hb A2 levels is important to diagnose the beta thalassemia trait (BTT). In some cases, the level of HbA2 is not typically elevated and some difficulties may arise in making the diagnosis and hence accurate assessment of HbA2 becomes of paramount importance. HPLC and HbCZE are considered acceptable methods to diagnose BTT but these vary in their accuracy and cut-offs. Complex elution patterns as well as co-elution of variants like HbA2, HbE, Hb Lepore, HbD Iran create difficulty in interpretation of HPLC plots.The aim of this study was to compare the results of HPLC and HbCZE technique for screening of clinically significant haemoglobin disorders. On comparing the HbA2 values the HPLC showed higher values for HbA2 with a median values as compared to HbCZE while when no variant was present, the Hb A2 concentrations showed excellent agreement. The Hb F, HbS measurement agreement was good between both methods. To conclude HPLC and HbCZE are complementary techniques and normal ranges and means normal values differ between different methods and different manufacturers and therefore it is important that each lab establish its own cut offs.","manuscriptTitle":"Diagnosis of Hemoglobinopathies and thalassemias using both Haemoglobin capillary zone electrophoresis and High performance liquid chromatography - An experience from a tertiary care hospital in Northern India","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-12 18:22:45","doi":"10.21203/rs.3.rs-4138194/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5a3dbfac-d28a-439e-b612-0aed3691be3e","owner":[],"postedDate":"April 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-12T18:27:56+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-12 18:22:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4138194","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4138194","identity":"rs-4138194","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}