| Abstract|| |
Background: Hemoglobinopathies are the most common inherited red cell disorders worldwide. Identification of these disorders is immensely important epidemiologically and for improved management protocols. Aim and Objectives: Our aim was to determine the prevalence of hemoglobinopathies in patients with microcytic hypochromic anemia and to assess the suitability of using high performance liquid chromatography (HPLC) routinely for screening antenatal cases and patients with anemia. Materials and Methods: A total of 4335 cases received from Mar 2007 to Nov 2011 were studied for various hemoglobinopathies and variants on BIO RAD 'VARIANT' analyzer. Results: Of the 4335 cases studied, 2119 were antenatal cases, 1710 patients with other disorders and 506 family studies. Of these, 688 cases displayed abnormal hemoglobin fractions on HPLC of which 140 were antenatal women. There were 455 cases of β thalassemia trait, 24 β thalassemia major, 20 thalassemia inter-media, 54 sickle cell trait, fivesickle cell disease, 21 double heterozygous β thalassemia-sickle cell trait, nineand 4 Hb D- Punjab heterozygous and homozygous respectively, three Hb D β Thalassemia trait, 20 and 37 Hb E homozygous and heterozygous respectively, three Hb E β Thalassemia trait and four cases of Hb Q India. Twenty nine adults had isolated HbF elevation. Conclusion: Our study found a high prevalence (15.8%) of hemoglobinopathies amongst microcytic hypochromic anemia and antenatal cases. An accurate diagnosis helps in preventing unnecessary iron loading. Screening all antenatal cases with anemia helps in timely antenatal counseling, thus preventing the psychological trauma of bearing a transfusion dependent child for life.
Keywords: Antenatal screening, hemoglobinopathies, high performance liquid chromatography, microcytic hypochromic anemia
|How to cite this article:|
Philip J, Sarkar RS, Kushwaha N. Microcytic hypochromic anemia: Should high performance liquid chromatography be used routinely for screening anemic and antenatal patients?. Indian J Pathol Microbiol 2013;56:109-13
|How to cite this URL:|
Philip J, Sarkar RS, Kushwaha N. Microcytic hypochromic anemia: Should high performance liquid chromatography be used routinely for screening anemic and antenatal patients?. Indian J Pathol Microbiol [serial online] 2013 [cited 2020 Nov 30];56:109-13. Available from: https://www.ijpmonline.org/text.asp?2013/56/2/109/118699
| Introduction|| |
Hemoglobin mutations form the most common human single gene disorders. About 698 genetically different hemoglobin variants are found scattered all over the world causing various hemoglobinopathies. About 5.2% of the world population (and more than 7% of pregnant females) carry a significant variant. About 1.1% of couples around the world are at risk for having children with a hemoglobin disorder of which 2.7 per 1000 conceptions are actually affected. Hemoglobin disorders contribute to 3.4% of mortality in children aged less than five years worldwide. 
Among these disorders, sickle cell syndromes and thalassemia constitute major public health problems. The frequency of beta-thalassemia trait (βTT) in India has been reported to vary from <1 to 17% depending on the region studied, with an average of 3.3%.  The average frequency of sickle cell disease is 4.3%.  Hemoglobin E has been reported as the most common hemoglobin (Hb) variant in Southeast Asia and the second most prevalent worldwide.  However in India, although common in the north-eastern region with a prevalence of 10.9% it is uncommon in other parts. 
The clinical manifestations of these hemoglobinopathies can vary from asymptomatic states to severe, lifelong, transfusion-dependent anemia with multi organ involvement and severely reduced life expectancy. Identification of these disorders is immensely important epidemiologically and to aid in prevention of the psychological and economic burden associated with a lifelong disorder. The aim of this study was to determine the prevalence of hemoglobinopathies in patients of anemia and to assess the suitability of using HPLC routinely for screening anemic and antenatal patients.
| Materials and Methods|| |
A total of 4335 cases received from Mar 2007 till Nov 2011 for hemoglobin (Hb) variant analysis were studied for various hemoglobinopathies and variants. The tests were performed on BIO RAD 'VARIANT' using beta thalassemia short program, (Bio-Rad Laboratories, California, USA). The instrument utilizes the principle of high performance liquid chromatography (HPLC). With HPLC, the positively charged Hb fractions are separated based on their ionic interactions with a negatively charged stationary phase in a chromatography column, followed by their elution by a mobile phase with phosphate buffers differing in pH and ionic strength. The adsorbed positively charged hemoglobin molecules are eluted from the column into the liquid phase at a rate related to their affinity for the stationary phase. Hemoglobins are identified by their retention time and quantified by computing the area under thecorresponding peak in the elution profile.  Hb A2 values ranging from 2.0 to 3.9%, and the Hb F values up to 1.3% were considered normal, which were provided by the manufacturer. The hemoglobins fall into windows which are defined by their retention times. Hemoglobins with retention times outside the windows are detected as unknown peaks.
Patients included cases of microcytic hypochromic anemia, referred to this centre where a co-existent hemoglobinopathy was suspected, while other cases were of anemia with no apparent underlying cause, antenatal cases, transfusion requiring children and adults and their family members.
About 2 ml of blood sample was collected in EDTA vial. They were stored at 4-8°C and were analyzed in batches once a week. Patients referred to us either already had a hemogram and a peripheral blood smear (PBS) reported or it was concurrently asked for. Concurrent iron deficiency anemia (IDA) was ruled out on the basis of iron studies including serum iron levels, total iron binding capacity, transferrin saturation and serum ferritin levels. Samples with abnormal results were also run on KX-21 hematologyanalyzer (Sysmex corporation, Kobe, Japan) to obtain hemoglobin values and indices. Sickling test was performed using freshly prepared sodium meta bisulphite when S-window was eluted in the sample. Other confirmatory tests including acid and alkaline electrophoresis were done when required.
| Results|| |
A total of 4335 cases were studied. The patient population comprised of 2119 (48.8%) antenatal patients, 1710 (39.4%) patients with other hematological or non hematological disorders and 506 (11.6%) family studies [Figure 1]. The age and gender wise distribution of patient population is shown in [Figure 2]. Of these, 688 (15.8%) cases displayed abnormal hemoglobin fractions on HPLC, 140 (20.3%) of which were in antenatal women. The major abnormality observed was of high Hb A2. A cut-off of over 3.9% was taken for diagnosis of βTT. Distribution of various hemoglobinopathies is as shown in [Table 1], [Figure 3]. The haemoglobin values and RBC indices in different haemoglobinopathies is as shown in [Table 2]. Twenty nine adult cases had isolated Hb F elevation of which eight were antenatal females. Hb F elevation could have been because of pregnancy per se or due to hereditary persistence of fetal hemoglobin (HPFH). A recommendation for molecular confirmation was given in these cases Distribution of hemoglobinopathies among antenatal females is shown in [Figure 4].
|Table 1: Hb fractions on high performance liquid chromatography in various hemoglobinopathies|
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|Figure 3: The distribution of hemoglobinopathies among positive cases Total no. of cases analyzed (n) = 4335 ,Pati ents with hemoglobinopathies (n) = 688 (15.8%)|
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|Figure 4: Distribution of hemoglobinopathies among antenatal patients Total number of patients (n) = 4335, Antenatal patients (n) = 2119, Antenatal patients with hemoglobinopathies (n) = 140 (6.6%)|
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| Discussion|| |
In our study we found that 688 (15.8%) of the patients with anemia had hemoglobinopathies. The prevalence of hemoglobinopathies among anemia patients varied in different studies depending on the region studied. In the study done by Sachdev et al. incidence of hemoglobinopathies was found to be 12.5% in the north Indian population.  Balgir found a prevalence of 65.7% in the state of Orissa.  Prevalence rate of 7% was seen in district Bhopal having mixed population of diverse ethnic groups and 16% in Maharashtra. , Since our study was conducted in Pune, Maharashtra it corroborates with the earlier findings. 
If the distribution of different hemoglobinopathies is analyzed, β TT was found to be the commonest disorder in most studies. ,,,, Sickle cell trait was the commonest disorder in Orissa.  The incidence of hemoglobinopathies in antenatal patients was 2.8% in the study by Sachdev et al.  whereas in our study it was found to be 6.6% possibly due to higher overall incidence of hemoglobinopathies in this region as compared to north India. , Among the antenatal population also, the commonest disorder was βTT.
The importance of careful and detailed complete blood count and peripheral blood smear (PBS) examination cannot be understated in the diagnosis of various hemoglobin disorders. In an α-thalassemia trait when the HPLC can be absolutely normal with perhaps a low normal or low Hb A2, careful correlation of the microcytosis which is unexplained by βTT or iron deficiency can lead us to suspect α-thalassemia disorder which can be confirmed by DNA analysis. All antenatal patients routinely undergo Hb investigation during their antenatal workup. Those with a low Hb are sent for PBS examination. Low mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) with a raised total RBC count is a pointer towards βTT. The PBS may show basophilic stippling and target cells which are more characteristic of β-thalassemia than iron deficiency anemia. Iron stores are normal/high in β-thalassemia. In an antenatal patient a strong possibility of α- thalassemia; if MCH is <27pg, and α0 thalassemia if MCH is <25 pg should be raised, especially if no Hb variant/βTT is detected on HPLC.  The Hb concentration in sickle cell trait is usually normal but in our study there were few patients with mild anemia. A co-existent iron deficiency anemia or an α-thalassemia trait needs to be ruled out in such cases. The PBS in sickle cell disorders will show sickle cells and the diagnosis can be confirmed by HPLC and induction of sickling by sodium metabisulphite. In other hemoglobinopathies the blood picture can be non-specific and HPLC comes in as an excellent diagnostic tool for detection and quantification of several normal and abnormal hemoglobins. Alternative techniques like acid and alkaline electrophoresis and IEF are also being used in various labs. Alkaline electrophoresis is rapid, reproducible, and capable of separating common hemoglobin variants, such as hemoglobin A (HbA), HbF, HbS, and HbC, but HbS, HbD, HbG, and HbLepore are unresolved from each other, as are HbC, HbA 2 , HbO-Arab, and HbE. In addition, there are other variants with electrophoretic mobilities identical or similar to those of HbS and HbC. Consequently, acid electrophoresis is needed for the identification of the aforementioned variants. Nevertheless, these electrophoretic methods are still not able, in most cases, to separate HbD from HbG and HbLepore and, in some cases, HbE from HbO-Arab. In Isoelectric focusing (IEF) very small differences in electrophoretic mobility can be picked up which help to resolve Hb C from Hb E and Hb S from Hb D as very sharp bands which makes quantization more accurate. However hemoglobins that can be distinguished from each other by IEF differ between different instrument/reagent systems. The precision at low concentrations is poor and this method is therefore not suitable for the quantification of hemoglobin A2. IEF is a more expensive procedure than electrophoresis on cellulose acetate, both because of greater capital costs and because the cost per test is greater. , Comparing various techniques, HPLC with its superior resolution, rapid assay time, and accurate quantification of hemoglobin fractions is an ideal methodology for the routine clinical laboratory. Although HPLC is a highly reliable technique, considerable expertise is required to interpret the data produced. Many hemoglobins may have same retention times as normal hemoglobins or other variants and co-inheritance of different traits can further confuse the issue. In our study the average Hb A2 in βTT was 4.8% versus 5.4% in South Indian subjects.  Cases of borderline Hb A2 values between 3 and 3.9% should be carefully analyzed as concomitant iron deficiency can lead to a low Hb A2 and mask a βTT. Timely detection of the trait in the antenatal patient and her spouse is important to prevent the birth of a homozygous thalassemia major child. A child with thalassemia major is dependent on regular blood transfusions to maintain life since early childhood. However safe blood is available only for a small fraction and most transfusion dependent patients die from iron overload unless an oral, inexpensive iron chelation is made more widely available.  In case a trait is diagnosed in an unmarried individual, genetic counselling regarding the nature of the disease and the implications of being carriers should be emphasized/explained.
We found 54 (1.24%) cases of sickle cell trait, 21 (0.48%) HbS/β-thal and 5 (0.11%) cases of sickle cell disease. Timely detection of sickle cell trait can be useful in warning patients of the possible complications and the preventive measures to be taken. Pre-natal or early post-natal diagnosis of homozygous sickle cell disease helps in institution of prompt therapy before the onset of serious complications of the disease.
Prevalence of another β chain variant Hb E in our study was: HbE-trait37 (0.85%) HbE/β-thal 03 (0.06%) Homozygous HbE disease 20 (0.46%) which is comparable to that found in other studies. , However the prevalence is high in north eastern states.  Identification of this Hb variant is important, because the doubly heterozygous state for HbE and β-thalassemia is characterized clinically by thalassemia major. It is important to increase the awareness of this relatively rare disorder, in clinicians and patients alike so as to assist in prenatal diagnosis, genetic counseling and clinical management.
Hemoglobin D Punjab variant shows a prevalence of 2-3% in Punjab, 1% in Gujaratis, 0.37% in Bengal with an overall prevalence of 0.86% in the general population. ,, Our study found 09 (0.20%) cases of HbD-Punjab trait as compared to 0.5% in north Indian population, 0.19% in Orissa, 0.15% in Maharashtra and 0.46% in Gujarat. ,,,
We also found04 (0.09%) homozygous HbD-Punjab and 03 (0.06%) cases of HbD/β-thal. Heterozygous Hb D disease is of no clinical significance. When associated with HbS it can cause a disorder similar to, but less severe than, sickle cell anemia. Homozygous Hb D disease causes mild hemolyticanemia and co-inheritance of β° thalassemia produces a mild thalassemic condition. The association between Hb D and hematological malignancies has also been reported. 
Hb Q India and HPFH are not of much clinical significance.
The correct and timely diagnosis of various hemoglobinopathies especially thalassemia, sickle cell anemia and their heterozygous combinations with other hemoglobins can help in institution of correct treatment to affected individuals, prevention of birth of homozygous affected individuals, genetic counseling to prevent further affected births, contain the morbidity, mortality psychosocial and financial burden placed on the families of affected individuals and the country as a whole.
In a study done in Hamilton  it was found that 39% patients with thalassemia trait were given iron therapy.  This can lead to unnecessary iron overload in such patients which would be harmful in the long run. In India premarital screening is still considered taboo. Hence the best approach would be to target those patients attending the hematology OPD, the antenatal population and extended family members (EFM) of known thalassemics/other hemoglobinopathies. In a study done by Tamhankar et al the yield of carriers from the EFM and OPD groups was 78.17 and 19.5% respectively. They could prevent the birth of 28 thalassemic childrenby screening of 394 individuals from the EFM group.  Screening can be extended to pregnant woman/individuals from a high-risk ethnic background and consanguineous marriages. The couple at risk should be counselled regarding the nature of the disease and the implications of being carriers. Options concerning birth control, including prenatal diagnosis and medical termination of pregnancy of the affected fetus should be given. Parents with affected children should be informed of 25% recurrence risk and advised to limit family size.
In a study conducted in Israel it was estimated that the lifetime cost of healthcare, early mortality, and lost earnings versus a national screening program including antenatal diagnosis gives a cost-benefit ratio of 4.22:1 and adding a social perspective 6.01:1. ,
Reviewing the data around the world especially in countries where the prevalence of thalassemia was high, we find that with effective population and antenatal screening programs these countries have been able to reduce the prevalence of hemoglobinopathies drastically. ,,, In a developing country like India with limited resources, a combined approach of primary and secondary prevention needs to be followed. Education and awareness regarding hemoglobinopathies needs to be spread among medical faculty, paramedics and general population. Secondly since population screening programs can be a Herculean task, financially impractical in a populous country like India, mandatory screening of antenatal cases, anemia patients and extended family members can be routinely done as it will prove to be cost effective considering the number of homozygous births which can be prevented vis-à-vis the financial burden on the health care system.
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Department of Transfusion Medicine, Armed Forces Medical College, Pune, Maharashtra
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]