Use of NESTROFT as a screening test for the carrier of Thalassemia major

Sharma G 1, Sharma D2 , Gulati R.K.3

1Dr. Gopikishan Sharma, PG Resident, Department of Pediatrics, Government medical College Kota, Rajasthan (India), 2Dr. Dipendra Sharma, Clinical Tutor and Junior Specialist, dept. of Pediatrics, GMC KOTA, 3Dr. R.K.Gulati, Senior Professor and Head of Department, dept. of Pediatrics, GMC KOTA.

Address for correspondence: Dr. Gopikishan Sharma, Email: dr.gopikishansharma6162@gmail.com


Abstract

Objective: To evaluate use of NESTROFT as a screening test for the carrier of thalassemia major. Design: prospective study. Settings and participants: 121 random clinically normal children who were  siblings of thalassemia major patients who registered at thalassemia welfare society of J K LON hospital kota. Intervention: All cases were investigated for CBC, NESTROFT and HbA2 estimation by HPLC method. Result: Total 121 cases were screened for carrier by HPLC method for HbA2 estimation. Total 59 (48.76%) cases had HbA2 level ≥3.5 %, considered as carrier while 62 (51.24%) cases were noncarrier. The Sensitivity, specificity, PPV and NPV of MCV<80, Mentzer index (<13 were  positive), and NESTORFT, were 89.83%, 46.77,61.62% ,82.85% ;  83.05%, 97.36%,  96.07% ,88.09 % and  93.22%, 88.70,  88.7 and 93.22 % respectively. So Nestorft is better test (sensitivity-93.22% and NPV is 93.22%) for screening for carrier. Conclusion: It is concluded that β thalassemia carriers are more prevalent in siblings of thalassemia major. For screening NESTORF test is reliable and cost effective and can be used for mass hemoglobinopathies screening programmes.

Keywords: Thalassemia carrier screening, HbA2 estimation, NESTROFT
 

Manuscript received: 16th May 2016, Reviewed: 26th May 2016
Author Corrected; 12th June 2016, Accepted for Publication: 25th June 2016

Introduction

In India β-thalassemia is the most common monogenic disorder. The average incidence of β-thalassemia trait in India is 3.3% with 1-2 per 1,000 couple being at risk of having an affected offspring each year. Prevalence of thalassemia trait varies form 1.0-14.9% in various regions of India. If we draw a line between Mumbai and Kolkata on the Indian map, in the region above the line the incidence of Thalassemia minor is higher (3-17%), where as in the region below the line incidence is less than  3%. Incidence varies in different communities, religions and ethnic group [1].

It is estimated that more than 25 million people in India, are carriers of the β-thalassemia gene and 8000 children are born every year with thalassemia major [1].  Only 10 to 15% of these children receive optimal treatment; [3] the cost of such treatment for one thalassemic child amounts to Rs. 90,000 to 1,00,000 annually at around 3 years of age, which increases as the child grows [4].

The only cure available today is bone marrow transplantation, which is not affordable to almost all patients in India. The birth of a thalassemic child, thus, places considerable physical, physiological and economic burden, not only on the affected child and its family, but also on the community and the nation at large. With these limitations, along with the treatment, measure for prevention of such births in the future should be undertaken [5]. Community control of hemoglobinopathies relies mainly on outreach educational programmes and genetic counselling with antenatal diagnosis [6]. Accurate and timely detection of various hemoglobin variant including β-thalassemia trait can prevent occurrence of more serious disorder like thalassemia major in newborn [2]. Various methods are there for screening of Thalassemia carrier. These are:-

Red cell indices- Thalassemic traits in general have reduced mean corpuscular volume (MCV) and reduced mean corpuscular hemoglobin (MCH) with normal mean corpuscular hemoglobin concentration (MCHC). Specific cut off points for each index varies from laboratory to laboratory. Some laboratories concentrate on both reduced MCV and MCH and some on MCV or MCH alone. Low MCV or MCH sometimes poses a problem by giving false positive results due to iron deficiency anemia or other nonthalassemic microcytosis.

Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT): NESTROFT is a rapid, simple and cost effective screening test. The principle of NESTROFT is based on the limit of hypotonicity on which the red cell can withstand. The use of NESTROFT has been recommended for mass screening due to its low cost and simplicity. Though NESTROFT is a simple and rapid test, combination of NESTROFT and red cell indices increases the sensitivity and negative predictive value to almost 100 per cent.

HbA2 determination--The hallmark of diagnosis for classical β thalassemia carriers is a raised HbA2 varying between 3.5 % and 4% depending on the method of estimation used.

In the present study we evaluated the efficacy of one such test, NESTROFT (Naked Eye Single Tube Red Cell Osmotic Fraglity Test) in comparison with various other screening parameters in the siblings of thalassemia major patients.
 
Material and Method

This prospective study was conducted in the department of Paediatrics, J. K. Lon Mother and Child Hospital, Government Medical College, Kota during the period of December 2013 to November 2014. All children who are siblings of thalassemia major patients registered at thalassemia welfare society at J.K. Lon hospital, Kota were included in the study. Total 121 cases were enrolled in the study.

Careful history was taken, all cases who had history of blood transfusion in last one month and acute febrile illness were excluded from our study. Family history was taken for consanguineous marriage, hemolytic anemia and other significant illnesses. General physical examination including vitals and anthropometry were taken followed by systemic examination. After that 5 ml blood was collected and sample was sent for following investigations:-

1. Complete Blood Count : Hemoglobin, RBC count, Hematocrit ,Red cell distribution width, MCV, MCH, MCHC, WBC counts, Platelet counts.

2. NESTROFT (Naked eye single tube red cell osmotic fragility test): This is used to assess osmotic fragility of red cells at a single concentration of buffered saline (0.36% in single tube) visually without a spectrophotometer.

A stock solution of 10% buffered saline (pH 7.4) was prepared by taking   NaCl----90g,    Na2HPO4 ---13.655 g,   NaH2PO4, 2H2O--- 2.4 g and dissolving them in a litre of distilled water. From this 0.36% saline prepared by dilution.

A positive NESTROFT indicates that all red cells in the tested sample have not undergone lysis in 0.36% buffered saline. These unlysed red cells result in the hazy appearance of the contents of the tube and render the line on the paper indistinct. Thus a positive NESTROFT indicates decreased red cell osmotic fragility and increased resistance to osmotic lysis.

3. High performance liquid chromatography (HPLC):- for HbA2 estimation Data obtained were tabulated using version 17 of the Statistical Package for Social Sciences (SPSS, published SPSS Inc.) and subjected to appropriate statistical analysis.
 
Result:-

Total 121 cases were screened for carrier of Thallsemia Major by HPLC method for HbA2 estimation. Total 59 (48.76%) cases had HbA2 level ≥3.5 %, considered as carrier while 62 (51.24%) cases were no carrier. Mean HbA2 value in carrier was 5.24% and in no carrier it was 2.69%. Difference was statistically significant(p<0.0001).

The mean MCV in carrier was 62.08 fl which was lower than the mean MCV in noncarrier (79.7fl) .The difference is statistically significant. MCV is key indicator for diagnosis and screening. The mean MCH in carriers of hemoglobinopathies was 21.35 pg which was lower than  noncarrier 25.60 pg (p value<0.001). A low MCH and a low MCV give a clue to the presence of thalassemia. [10]. In this study the mean MCHC in carriers of hemoglobinopathies was 31.55 % which was lower than noncarrier 31.65% and consistant with known literature [8].

In our study 86 cases had MCV<80. Out of them 53 cases were carrier and 33 cases were noncarrier. 6 carrier had MCV >80 and 29 noncarrier had MCV>80. In our study the Sensitivity, specificity, PPV and NPV of MCV<80 is 89.83%, 61.62%, 82.85% respectively. All the parameters are statistically significant.(p value- <0.0001). In our study we calculated Mentzer index (TRBC/MCV) of the patient. This index was <13 considered positive for screening of carrier. The Sensitivity, Specificity, PPV and NPV of Mentzer index are 83.05%, 97.36%, 96.07% and 88.09 % respectively in current study. All the parameters are statistically significant (p value<0.001).

Nestorf test was done in each case. Cases having Nestorf test positive were 62. Out of them 55cases were carrier and 7 cases were noncarrier. 4 carriers had Nestorf test negative and 55 noncarrier had Nestorf negative.

In our study the Sensitivity of Nestorf test positive as screening of thalassemia carrier was 93.22%, specificity was 88.70%, % false positive cases were 5.78% ,   % false negative cases are 3.3%,  predictive value of positive test was 88.7% and Predictive value of  negative test was 93.22%. All the parameters were statistically significant and p value was <0.0001

Discussion

Preventive screening programmes to identify carriers are being used by many countries where thalassemia is a common disease. Our study is done to evaluate the use of NESTORFT for mass screening. In our study total 121 cases were screened for carrier of Thallesemia Major by HPLC method for HbA2 estimation. Total 59 cases had HbA2 level >3.5 % considered as carrier which are 48.76% of total. In our study we considered carrier as HbA2 ≥3.5%.

The mean MCV in carrier was 62.08 fl which was lower than the mean MCV in noncarrier (79.7fl). The difference is statistically significant. MCV is key indicator for diagnosis and screening. Thalassemic individuals have a reduced MCV, and one study has suggested that an MCV of <72 is maximally sensitive and specific for presumptive diagnosis of thalassemia syndromes. [11,15].

The mean MCH in carriers of hemoglobinopathies was 21.35 pg which was lower than  noncarrier 25.60 pg ( difference is statistically significant-p value<0.001) . A low MCH and a low MCV give a clue to the presence of thalassemia [10].

As per result 86 cases had MCV<80. Out of them 53 cases were carrier and 33 cases were noncarrier. 6 carrier had MCV >80 and 29 noncarrier had MCV>80. In our study the Sensitivity, specificity, PPV and NPV of MCV<80 is 89.83%, 61.62%, 82.85% respectively. All the parameters are statistically significant. Maheshwari et al reported in 1286 cases sensitivity and specificity of both MCV(<77 fl) and MCH(<27pg) was 98% and 92%. The values were higher than our study because of selection of two parameters MCV and MCH [14]. Mangalani et al reorted MCV<80fl  sensitivity -93.7%, specificity- 40.6%, negative predictive value- 95.8% and positive predictive value - 32.5% in 2525 cases [13]. BC Mehta et al(2002) reported sensitivity of MCV <77 fl was97.4%, specificity -29.7%, NPV -97.5% and PPV-28.2%. [7], Batebi et al (2012)  reported sensitivity of MCV was 81.3 %, specificity 81.7%, PPV-81.1%, NPV-94.9%.

As above in result we used Mentzer index (TRBC/MCV) for screening of thalassemia carrier. This index was <13 considerd positive. The Sensitivity, Specificity, PPV and NPV of Mentzer index are 83.05%, 97.36%, 96.07% and 88.09% respectively in current study. All the parameters are statistically significant (p value<0.001). The Sensitivity of Mentzer index was 84.6% in George Klee et al study [9], 88.7% in Nishi madan et al study [16], and 85.3% in sreekantha et al [12].

In our study Nestroft done in all cases, the Sensitivity of Nestorf test is 93.22%,specificity is 88.70%, predictive value of positive test is 88.7% and Predictive value of  negative test is 93.22%. All the parameters are statistically significant and p value is <.0001.So as per table I the result of sensitivity are comparable to Mamta et al and Maheshwari et al [14,15], specificity is comparable to Raghvan et al and Singh et al [17,18], PPV is comparable to Thomas et al and NPV is slightly lower than Thomas et al while higher than Singh et al [17] . But negative predictive value is more than 90% in all above study as also in our study.

As per table II sensitivity & NPV of Nestorf test is higher than all other methods of screening in our study, which is also present in all other studies. Due to higher sensitivity and negative predictive values, NESTROFT can be used as  mass screening test.

Conclusion

It is concluded that β thalassemia carriers are more prevalent in siblings of thalassemia major. Hemoglobinopathies can be suspected on the basis of hematological parameters like reduced MCV, reduced MCH, and elevated RBC count disproportionate to hemoglobin level.  NESTORF test is reliable, cost effective and better screening test for carrier and positive cases can be confirmed by HPLC. We recommend to screen the carriers and counsel them to screen the forecoming lifepartener for thalassemia carrier before marriage to prevent thalassemia major in their next generation. For screening we recommended NESTORF test and also can be used in mass hemoglobinopathies screening programmes.

Table I:-Comparative study for NESTROFT in various studies

Study

No. of cases

Sensitivity

Specificity

PPV

NPV

Manjulata etal(1999) [14]

1048

91%

95%

55%

99%

Mamta manglani etal(1997) [13]

1695

94.4%

64.2%

35.3%

97.6%

Mehta et al(1991) [10]

131

99.2%

75.8%

69.3%

99.5%

Thomas et al(1996) [17]

137

98.7%

66.6%

87%

96.5%

Raghvan et al(1991) [18]

-

95.5%

87%

70.5%

98.3%

Present study

121

93.22

88.7

88.7

93.22

Table- II:-Comparision of  Nestorf  test  with other screening test

Name of test

Sensitivity

Specificity

PPV

NPV

MCV<80

89.83%

46.77%

61.62%

82.85%

Mentzer test

83.05%

97.36%

96.07%

88.09%

Nestorf test

93.22%

88.7%

88.7%

93.22%

Funding: Nil, Conflict of interest: Nil    
Permission from IRB: Yes

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How to cite this article?

Sharma G.K, Sharma D, Gulati R.K. Use of NESTROFT as a screening test for the carriers of thalassemia major. Int J Pediatr Res.2016;3(7):502-506.doi:10.17511/ijpr.2016.7.06