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ORIGINAL ARTICLE
Year : 2013  |  Volume : 6  |  Issue : 1  |  Page : 71-74  

Screening of β-thalassaemia trait by means of red cell indices and derived formulae


1 Department of Pathology and Microbiology, Sree Narayana Institue of Medical Sciences, Ernakulam, India
2 Department of Pathology and Microbiology, Mahatma Gandhi Medical College, Jaipur, India

Date of Web Publication14-Mar-2013

Correspondence Address:
Shashikant Adlekha
Sree Narayana Institute of Medical Sciences, Chalakka, North Kuthiyatode, P.O. 683594, Dist.: Ernakulam, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0975-2870.108649

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  Abstract 

Context: Thalassaemias are a group of genetic disorders with tremendous morbidity and mortality that are present widely across the globe. Aim of the Study: To evaluate the usefulness of automated haematology analyser-based red cell indices and formulae derived from these indices in β-thalassaemia trait (BTT) detection. Settings and Design: Prospective study conducted over a period of 1 year. Materials and Methods: The study included 301 cases with mild-to-moderate reduction in haemoglobin (Hb) with a lower cut-off value of 8 g/dl. Automated haematology analysis was done by using an "Arcus Diatron" (2000) for all these cases. A cut-off value of mean cell volume (MCV) < 76 fl and serum ferritin level < 12 ng/ml were taken for high-performance liquid hromatography (HPLC) screening. Based on these indices we evaluated eight automated analyser red cell indices and formulae - red blood cell count (RBCC), MCV, mean cell Hb (MCH), red cell distribution width (RDW), red cell distribution width index (RDWI), Mentzer index (MI), discriminant score (DS) and mean density of Hb per liter (MDHL) - for all 301 cases. Youden's index was computed for all derived indices. Results : Out of 98 cases screened by HPLC (D-10 VARIANT; Bio-Rad Laboratories), 88 turned out to be positive for BTT (HbA2 > 3.5%). Indices like RDW, RDW with low MCV, MCH, RDWI and DS were found to have a better discriminating function (higher Youden's index) compared with RBCC, MDHL and MI. Conclusion: Automated haematology analyser-based red cell indices and certain formulae derived from these indices have a high predictive value in screening BTT.

Keywords: β-Thalassaemia trait, haematology analyser, HbA2, HPLC, iron deficiency anaemia


How to cite this article:
Adlekha S, Chadha T, Jaiswal RM, Singla A. Screening of β-thalassaemia trait by means of red cell indices and derived formulae. Med J DY Patil Univ 2013;6:71-4

How to cite this URL:
Adlekha S, Chadha T, Jaiswal RM, Singla A. Screening of β-thalassaemia trait by means of red cell indices and derived formulae. Med J DY Patil Univ [serial online] 2013 [cited 2024 Mar 29];6:71-4. Available from: https://journals.lww.com/mjdy/pages/default.aspx/text.asp?2013/6/1/71/108649


  Introduction Top


Thalassaemias are a heterogeneous group of disorders with a genetically determined reduction in the synthesis of one or more type of polypeptide chains of normal haemoglobin (Hb). They are the commonest monogenic disorders in man. There are two main types of thalassaemia, one affecting the synthesis of the α-chain and the other affecting the synthesis of the β-chain, called as α-thalassaemia and β-thalassaemia, respectively.

They occur at a high gene frequency through the Mediterranean population, the Middle East, the Indian subcontinent, Burma, and in a line stretching from southern China through Thailand and the Malay Peninsula into the island populations of the Pacific. [1] In Europe, they are particularly more in inhabitants of Italy and Greece. In Greece and Italy, there are an estimated 3500 and 6500 individuals, respectively, who are affected by β-thalassaemia major. [2] In India, the mean prevalence of β-thalassaemia trait is 3.3%. [3] A total of 6000-8000 children are born every year with β-thalassaemia major. [4]

Diagnosis of β-thalassaemia trait (BTT) often becomes perplexing due to its overlapping and similar features with iron deficiency anaemia (IDA). There are similarities in red cell indices between the two disorders [5] such as reduced Hb, MCV, MCH, and marked degree of microcytosis and hypo-chromia seen in both conditions in peripheral smears. Clinical features do not help much in their discrimination from each other. In usual cases of BTT, red cells exhibit various anomalies like microcytosis, hypo-chromia and reduction in MCV, which are relatively more marked, considering mild or absent anaemia. Conversely, in IDA, there is a closer correlation between the morphological abnormalities of red cells and the degree of anaemia. Red cell anisocytosis as measured by the cell sizing facility of some modern electronic haematology analysers is often more marked in iron deficiency.

Estimation of serum iron, ferritin, transferrin, HbA2 and HbF yields a definitive diagnosis of IDA and BTT. Homozygous thalassaemia can be prevented through detection and proper awareness of heterozygous carriers. One way of achieving this goal is to screen the population at risk. [6] Various electrophoretic approaches, including cellulose acetate electrophoresis, isoelectric focusing in polyacrylamide or agarose gel, as well as high-performance liquid chromatography (HPLC), immunological assays, structural analysis and genotype methods, are used to investigate haemoglobinopathies. Automated analysis of blood has made the red cell indices more accurate, reproducible and readily available even without experienced morphologists.


  Material and Methods Top


We prospectively analysed cases presenting at the Central Laboratory (both out-patient and in-patient) for Hb screening. Those with mild-to-moderate reduction in Hb level corresponding to their age group (adult males: 12.9- 8.0 g/dl, adult non-pregnant females: 11.9-8.0 g/dl, children up to 14-year age group: 11.9-8.0 g/dl) [7] were chosen for screening.

A lower limit of 8.0 g/dl was chosen as a criterion for screening, as the range of Hb in BTT patients is usually 9-12 g/dl. [1] Severe anaemia cases (< 8 g/dl) [7] are rarely confused with BTT in clinical practice, [8] so these cases were excluded in the present study. We chose 301 samples randomly from the males and non-pregnant females of the 1- to 60-year age group for initial screening using automated haematology analyser red cell indices. All samples were subjected to automated haematology analyser evaluation of various red cell indices and then for serum ferritin estimation. We adopted the following criteria: samples with MCV < 76 fl [9] and normal serum ferritin status (serum ferritin < 12 ng/ ml) [8] were further evaluated by HPLC. The criterion to label the cases as BTT was HbA2 > 3.5%. We excluded cases of BTT with concomitant IDA, which vary in certain parameters from classical BTT cases.

We computed various automated analyser red cell indices and their derived formulae:

MI = MCV/RBC [10]

MDHL = MCH/MCV × RBCC (× 10 12 /l), [11] MDHL > the median MDHL of the normal population signifies BTT; MDHL < the median MDHL of the normal population signifies IDA. In our study, we calculated the median MDHL of the normal population by taking samples from 20 males and 20 females with normal Hb, MCV, MCH and mean cell Hb concentration (MCHC), and computed median MDHL as 1.56 for males and 1.31 for females.

DS [11] = (0.096 × MCV) + (0.415 × RDW) - (0.139 × RBC)-12.722 for males;

(0.096 × MCV) + (0.415 × RDW) - 12.722 for females;

DS > 0.3095: IDA; DS < 0.3095: BTT.

Red cell distribution width (RDW) index (RDWI): [12] MCV × RDW/RBCC.

We evaluated sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Youden's index.

Sensitivity = True positive/ (True positive + False negative); Specificity = True negative/(True negative + False positive); PPV = True positive/(True positive + False positive); NPV = True negative/(True negative + False negative).

Youden's index = Sensitivity + Specificity - 100.

Statistical software SPSS 16 version was used for statistical analysis.

The three part automated haematology analyser used in the present study was, "Arcus Diatron", working on the principle of impedance. The three-level controls were run daily and the instrument was maintained according to the manufacturer's instructions. HbA2 estimation by HPLC was done using D-10 VARIANT (Bio-Rad Laboratories, Hercules, CA, USA). The instrument was maintained according to the manufacturer's instructions. For serum ferritin estimation, we used Randox Daytona, a fully automated biochemical analyser (RX Daytona-2005). The Randox was calibrated at a regular interval of 1 week by a calibrator, and the two- level controls were run daily and the instrument was maintained according to the manufacturer's instructions.


  Results Top


Out of the 301 cases screened initially by haematology analyser, 88 were of BTT [Table 1], 188 cases were of IDA and the rest 25 cases were non-IDA, non-BTT (anaemia due to other causes). This distinction into three groups was made on the basis of serum ferritin estimation and HbA2 estimation by HPLC. On comparison of mean Hb, RBCC, MCH, MCHC and RDW between the BTT and IDA group [Table 2], we found a significant difference (P < 0.05) between the two groups in all the red cell indices except MCHC. We subsequently evaluated the sensitivity, specificity, positive and negative predictive values and Youden's index [Table 3] of the RBCC and derived formulae - MI, DS, MDHL and RDWI - in discriminating BTT cases from other microcytic hypo-chromic anaemia cases. RDWI showed maximum Youden's index of 80 followed by DS with a Youden's index of 69, followed by MDHL, MI and RBCC in descending order.
Table 1: Screening results for HbA2

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Table 2: Laboratory values of individuals in the BTT and IDA groups
(mean ± SD)


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Table 3: Red cell indices in the diagnosis of BTT

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  Discussion Top


Thalassaemia is a widely prevalent disease that causes not only health hazard but also a tremendous socioeconomic burden on the affected, their family and the society. The current study provided a systematic approach of screening BTT, discriminating from IDA and thus minimising the incidence of the deadly homozygous thalassaemia. Decreased MCV with normal RDW status, when seen in non-nutritional deficiency anaemia cases, was found to be a highly reliable, convenient and cost-effective approach for mass screening of BTT. Low MCH count was significantly sensitive in diagnosing BTT, with all our BTT cases having MCH < 27. High RBCC, although found in a high number of BTT cases, its sensitivity in detecting BTT was very low (40.9%) and it cannot be effectively used to screen BTT cases. In our study, the RBCC of BTT cases was significantly elevated, with mean RBCC of 5.43 ± 0.97. It showed significant difference with the mean RBCC of IDA cases, which was 4.16 ± 0.82. This is in line with a similar observation laid by the studies of Bencaiova et al., [13] Reza et al. [14] and Beshlawy et al. [15] Newer red cell indices have emerged as effective discriminants between BTT and IDA, and are being potentially used for screening BTT. In our study, MI (MCV/RBC < 13) showed a sensitivity of 68%, specificity of 89% and PPV of 75%. A similar observation was made by Manglani et al., [16] with MI showing sensitivity of 66.2%, and Afroz et al. [17] showed MI having PPV of 91%. A sensitivity of 89% and specificity of 81% was observed for MI in a study by Niazi et al. [18]

In our study, MDHL showed sensitivity of 77.2% and specificity of 85.1%, and DS showed sensitivity of 95% and specificity of 74%. This is in contrast to the observations made by Telmissani et al. [11] in which MDHL showed sensitivity of 90% and specificity of 100%, whereas DS showed sensitivity of 83% and specificity of 81.4%. This can be attributed to the variations in the regional populations, resulting in a significant difference between the median MDHL of their and our study.

In our study, RDWI < 220 showed sensitivity of 91% and specificity of 89%. This is in accordance with the observations made by Demir et al., [12] showing sensitivity of 92% and specificity of 88%. Bencaiova et al. [13] showed that RDWI < 220 has sensitivity of 15.2% and specificity of 99.9%, and RDWI < 280 has sensitivity of 68% and specificity of 94%. A study by Nesa et al. [2] showed RDWI sensitivity and specificity of > 80% in the detection of BTT and IDA cases, and a Youden's index of 65.4. In the present study, the highest Youden's index was obtained for RDWI (80) followed by DS (69) and further in decreasing order by MDHL, MI and RBCC. Youden's index takes into account both sensitivity and specificity, and gives an appropriate measure of the validity of a technique. In the present study, Youden's index evaluated the discriminating function of the red cell indices and their derived formulae. Various other red cell indices such as England and Fraser, and Shine and Lal have been evaluated by studies such as the one by Batebi et al., [19] showing a high sensitivity of 87.2% for the England and Fraser index in the diagnosis of BTT, and thus, it was proposed as an acceptable discriminator between BTT and IDA. The discriminating function of the various indices is dependent on the age of a patient. A study by Rahim et al. [20] showed that the most accurate discriminant index for those younger than 10 years is RBCC and the Shine and Lal index, and for those older than 10 years is RBCC and RDW. The limitation of the present study was that, not all red cell indices were analysed. There is need of another single study evaluating the discriminating function of all possible red cell indices published in the literature so far.


  Conclusion Top


From the present study, we have come to the conclusion that with the easier availability of automation in haematology, the automated haematology analyser red cell indices have become fairly sensitive, specific, reproducible and precise. They can eliminate the need of further follow-up of cases by expensive investigations like electrophoresis and HPLC, and thus minimise the expenditure during mass screening of BTT. None of the red cell indices evaluated in our study had cent percent sensitivity and specificity in diagnosing BTT, although newer red cell indices like RDWI and DS were found to have high sensitivity and specificity in predicting BTT, and they can be effectively used in screening of BTT, as also suggested by other studies. Other newer red cell indices like MDHL and MI were found to have sensitivity of moderate range, and if combined with other sensitive parameters like low MCV and normal RDW, they can be effectively used for mass screening of BTT.

 
  References Top

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5.Rahim F, Saki N. Age-specific cutoff in discriminating iron deficiency anaemia from beta thalassemia traits. IJBC 2010;2:197.  Back to cited text no. 5
    
6.Shalev O, Yehezkel E, Rachmilewitz EA. Inadequate utilization of routine electronic RBC counts to identify β thalassaemia carriers. Am J Public Health 1993;78:1476-7.  Back to cited text no. 6
    
7.WHO. Haemoglobin concentrations for the diagnosis of anaemia and assessment of severity. Vitamin and Mineral Nutrition Information System, Geneva, World Health Organization, 2011 (WHO/NMH/NHD/MNM/11.1). Available from: http://www.who.int/vmnis/indicators/haemoglobin.pdf. [Last accessed on 2011 June 7].  Back to cited text no. 7
    
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10.Mentzer WC. Differentiation of iron deficiency from thalassaemia trait. Lancet 1973;1:882.  Back to cited text no. 10
    
11.Telmissani OA, Khalil S, Roberts GT. Mean density of hemoglobin per liter of blood: A new hematological parameter with an inherent discriminant function. Lab Hematol 1999;5:149-52.   Back to cited text no. 11
    
12.Demir A, Yarali N, Fisgin T, Duru F, Kara A. Most reliable indices in differentiation between thalassaemia trait and iron deficiency anemia. Pediatr Int 2002;44:612-6.   Back to cited text no. 12
    
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15.El-Beshlawy A, Kaddah N, Moustafa A, Mouktar G, Youssry I. Screening for β thalassaemia carriers in Egypt: Significance of the osmotic fragility test. East Mediterr Health J 2007;13:780-6.  Back to cited text no. 15
    
16.Manglani M, Lokeshwar MR, Vani VG, Bhatia N, Mhaskar V. Nestroft - An effective screening test for β thalassaemia trait. Indian Pediatr 1997;34:702-7.  Back to cited text no. 16
    
17.Afroz M, Shamsi TS, Syed S. Predictive value of MCV/RBC count ratio to discriminate between iron deficiency anemia and β thalassaemia trait. J Pak Med Assoc 1998;48:18-9.  Back to cited text no. 17
    
18.Niazi M, Tahir M, Raziq F, Hameed A. Usefulness of red cell indices in differentiating microcytic hypochromic anemias. Gomal J Med Sci 2010;8:125-9.  Back to cited text no. 18
    
19.Batebi A, Pourreza A, Esmailian R. Discrimination of β-thalassaemia minor and iron deficiency anemia by screening test for red blood cell indices. Turk J Med Sci 2012;42:275-80.  Back to cited text no. 19
    
20.Rahim F, Keikhaei B. Better differential diagnosis of iron deficiency anaemia from beta thalassemia trait. Turk J Haematol 2009;26:138-45.  Back to cited text no. 20
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3]


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