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 ¤  What is Nestroft?
 ¤  1.2 How is Nestr...
 ¤  1.3 How is Nestr...
 ¤  1.4 What does a ...
 ¤  2.1 What is the ...
 ¤  3.1 Why identify...
 ¤  4.1 What is the ...
 ¤  4.2 How does NES...
 ¤  5.1 Population s...
 ¤  5.2 The use of N...
 ¤  5.3 The use of N...
 ¤  References
 ¤  Article Tables

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Year : 2002  |  Volume : 56  |  Issue : 11  |  Page : 537-544

NESTROFT: A screening test for beta thalassemia trait

Dr J C Patel Dept. of Hematology, Seth G S Medical College & KEM Hospital, Mumbai 400012; Director, Laboratory Services and Blood Bank , BSES MG Hospital, Andheri, 400 058, India

Correspondence Address:
B C Mehta
Dr J C Patel Dept. of Hematology, Seth G S Medical College & KEM Hospital, Mumbai 400012; Director, Laboratory Services and Blood Bank , BSES MG Hospital, Andheri, 400 058
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Source of Support: None, Conflict of Interest: None

PMID: 14510336

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How to cite this article:
Mehta B C. NESTROFT: A screening test for beta thalassemia trait. Indian J Med Sci 2002;56:537-44

How to cite this URL:
Mehta B C. NESTROFT: A screening test for beta thalassemia trait. Indian J Med Sci [serial online] 2002 [cited 2016 May 28];56:537-44. Available from:

 ¤ What is Nestroft? Top

The acronym NESTROFT stands for naked eye single tube red cell osmotic fragility test. We have been evaluating this test in our laboratories since 1985, and the term NESTROFT was first used by us in 1988 in our initial paper descri­bing its use in population screening for beta thalassemia trait (BTT). [1] Effectiveness of this test in detecting BTT was shown in 1981. [2]

As its name implies, NESTROFT 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.

 ¤ 1.2 How is Nestroft Performed? Top

A stock solution of 10% buffered saline (pH 7.4) is prepared by taking NaCI 90g, Na 2 HPO 4 13.655 g and NaH 2 PO 4 , 2H 2 0 2.4 g, and dissolving them in a litre of distilled water. From this, a litre of 1 buffered saline is prepared by 1:10 dilu­tion with distilled water. 0.36% buffered saline is prepared by diluting 36 mL of 1 % buffered saline with 64 mL distilled water to make 100 mL.

2mL of 0.36% buffered saline is taken in one tube (10 cm x 1 cm diameter) and 2 mL distilled water is taken in another. A drop of blood is added to each of the tubes, which are left undisturbed for half an hour at room temperature.

 ¤ 1.3 How is Nestroft Interpre­ted?  Top
After half an hour the contents of both the tubes are shaken and the tubes held against a white paper on which a thin black line is drawn. The line is clearly visible through the contents of the tube containing distilled water due to com­plete lysis. If the line is visible through the contents of the tube with buffered sa­line, the test is negative, whereas if the line is not visible, the test is positive.

The tubes are then left undisturbed for a few hours. At the end of this period, con­tents of the tube with distilled water re­main uniformly pink with no sediment at the bottom. In the case of a negative test, the tube containing buffered saline also presents a similar picture. With a posi­tive test, the tube shows a sediment of the red cells at the bottom and the top part of the saline is colorless. This is an additional confirmatory evidence of a test earlier interpreted as positive.

 ¤ 1.4 What does a Positive Nestroft mean? Top

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. These red cells also sediment as a button at the bottom of the tube when it is left undisturbed for some time. Thus a positive NESTROFT indicates decreased red cell osmotic fragility and increased resistance to osmotic lysis.

 ¤ 2.1 What is the Utility of Nestroft? Top

Red cell osmotic fragility is decreased in BTT, resulting in a positive NESTROFT. NESTROFT is also positive in other conditions which are associated with decreased red cell osmotic fragility such as iron deficiency anemia (IDA), liver diseases [3] and hemoglobinopathies.

NESTROFT can be a useful test to screen the general population for BTT, and has been used for this purpose in India and in other countries [4],[5] where BTT is common.

 ¤ 3.1 Why identify individuals with Beta Thalassemia Trait? Top

3.1.1 Prevention of beta thalassemia major

Advances in molecular diagnostic tech­niques have made it possible to diagnose beta thalassemia major (BTM) at 6-10 weeks of gestation. [6] Early antenatal di­agnosis of BTM followed by medical ter­mination of pregnancy can prevent birth of children with BTM. Conventional therapy of BTM is life-long and places a significant load on blood transfusion services and finances. Prevention of birth of children with BTM would thus spare a lot of distress, effort and expenses for the families involved and for society. The key requirement for this is identification of couples at risk of giving birth to chil­dren with BTM. This can be done in two ways.

3.1.1. A Population screening for beta thalassemia trait

While screening of the general popula­tion would be the best way if resources were unlimited and the population man­ageable. In a populous country such as India screening of communities known to have high prevalence of BTT would be more cost-effective and practical. While conventional counseling would be to advise an individual with BTT not to marry another individual with BTT, we have found "positive counseling" to be more acceptable and extremely useful; i.e. an individual free of BTT can marry an individual with BTT without any risk of producing a child with BTM. Couples at risk of having children with BTM can be advised about the need for prenatal diagnosis at an early stage of pregnancy. If the exact mutation is identified before­hand, it can make prenatal diagnosis more certain. In the absence of appro­priate action following identification of individuals at risk of producing children with BTM, the purpose of screening pro­gram is not achieved and the cooperation of the target population can be lost.

3.1.1. B Antenatal screening

All pregnant women attending antena­tal clinics can be screened for BTT at the time of their first antenatal visit. The husbands of women who are found to be carriers after confirmatory tests should then be tested for BTT to iden­tify pregnancies at risk of producing chil­dren with BTM. This may not be useful for the index pregnancy if the first ante­natal visit is at 16-20 weeks of gesta­tion, but it would be useful for timely action during subsequent pregnancies. Women identified to have BTT should avoid the routine antenatal iron therapy as mentioned in 3.1.2 Family physicians, if they are made aware of screening pro­grams and their importance, can advise screening for BTT at an early stage of pregnancy.

3.1.2 Avoidance of unnecessary iron therapy

Mild to moderate degree of hypochro­mic-microcytic anemia is encountered in about 65-85% of carriers of BTT .[7],[8] In the absence of definitive diagnosis, many of these patients receive oral or parenteral iron therapy. This may go on intermit­tently or continuously for prolonged pe­riods of time because the anemia per­sists. [9] This sort of iron therapy is not only unnecessary but may be harmful be­cause of iron overload

3.1.3 Rational approach to hypochro­mic microcytic anemia

Hypochromic microcytic anemia due to iron deficiency, chronic diseases or BTT comprise almost 80% of the cases of anemia seen in India. It is necessary to distinguish between the three causes precisely since the therapeutic approach is different for each.

 ¤ 4.1 What is the need for a Screening test for BTT? Top

The diagnostic test for BTT is estima­tion of hemoglobin A2 by electrophore­sis or column chromatography. Both these techniques are expensive and time-consuming, and need expensive equipment and laboratory expertise. Be­cause of these limitations, these tests can only be carried out on a limited num­ber of samples and only in some labora­tories.

Cost-effectiveness can be improved if definitive tests were to be performed only on samples with high chances of yielding positive results. Universal use of definitive tests for population surveys or in antenatal clinics or in hematology clinics would not be acceptable due to the cost and effort involved. The avail­ability of an initial screening test which, while being inexpensive and easy to per­form even under field conditions, can identify positive samples with great sen­sitivity and reasonable specificity, would be technically and financially attractive.

 ¤ 4.2 How does NESTROFT compare with other screening tests for BTT? Top

Mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) are reduced in BTT as well as IDA, and can by themselves not be used to distin­guish between the two. Various discrimi­nant functions based upon red tell indi­ces, hemoglobin and red cell count have been devised to differentiate between BTT and IDA. [10],[11] Red cell indices can be accurately determined only with elec­tronic particle counters which are expen­sive, unsuited for field use, and need regular maintenance, standardization and quality control. Only a small number of laboratories in developing countries can satisfy all these requirements.

NESTROFT does not require any spe­cialized equipment, expertise or rigid conditions, and can be used in field con­ditions. NESTROFT campares favorably with MCV from the point of view of sen­sitivity, specificity and efficiency (see 5.1, 5.2, 5.3 and Appendix).

 ¤ 5.1 Population survey for BTT using NESTROFT Top

Individuals belonging to the Lohana community were screened for BTT NESTROFT, serum ferritin (Radio immunometric assay using Pharmacia kits) and MCV (Erma PC-604 cell counter) were determined in 450 indi­viduals. HbA2 was estimated (paper electrophoresis with elution [10] ) in 170 cases. This included all 61 individuals with positive NESTROFT, all 46 individu­als with negative NESTROFT and MCV <75 fL and 63 randomly selected indi­viduals with negative NESTROFT and MCV >75 fL. Serum ferritin of 10 mcg/L or less was considered suggestive of iron deficiency.

The results of the survey are shown in [Table 1],[Table 2] and [Table 3]. Both NESTROFT and MCV were highly sensitive (98%) for the detection of BTT, while the specificity of NESTROFT (86.6%) was more than that of MCV (66%). While the predictive value of a negative result was high (approxi­mately 99%) for both, the predictive value of a positive result was higher for NESTROFT (64.9%) than for MCV (42.1 %). NESTROFT was more efficient (88.9%) than MCV (72.4%) for screen­ing for BTT in this population survey.

 ¤ 5.2 The use of NESTROFT in Ante­natal Clinics Top

Twenty-three hundred fifty women at­tending the antenatal clinics of three hospitals or a number of nursing homes were surveyed. NESTROFT, was done in all 2350 women. MCV, serum ferritin and HbA2 were done in 449 cases. This included all 115 women with positive NESTROFT, all 200 women seen in nursing homes, all 150 women from one of the three hospitals, and 77 randomly selected women with negative NESTROFT. Results of the study are shown in [Table 4],[Table 5] and [Table 6]. It can be seen that NESTROFT is more sensitive (100%) and specific (87.7%) than MCV (91.4% and 57% respectively).

Predictive value of a negative test is high with both NESTROFT (100%) and MCV (98.2%). Predictive value of a positive test in yielding the diagnosis of BTT is higher with NESTROFT (59.1%) than with MCV (28.1%). In the present study of screening for BTT in antenatal women, NESTROFT was more efficient (89.5%) than MCV (49.5%).

 ¤ 5.3 The use of NESTROFT in Hematology Clinics Top

Twenty-five hundred consecutive pa­tients attending the hematology clinic of a teaching hospital or a private hema­tology clinic were studied. NESTROFT and MCV were done in all of them. HbA2 and transferrin saturation were estimated in 1196 cases. This included all 426 pa­tients with positive NESTROFT, all 493 patients with negative NESTROFT and MCV < 75 fL, and 284 randomly selected cases with negative NESTROFT and MCV >75 fL. The results are shown in [Table 7],[Table 8] and [Table 9]. It can be seen that the sensitivity of NESTROFT (99.2%) and MCV (97.4%) for detection of BTT was high. The specificity of NESTROFT was higher (80%) than that of MCV (29.7%). While predictive values of negative results were similar for NESTROFT (99.7%) and MCV (97.5%), the predictive value of a positive result with NESTROFT was higher (61.5%) than with MCV (28.2%). Efficiency of NESTROFT (86.1 %) was better than that of MCV (44.7%).

 ¤ References Top

1.Mehta BC, Gandhi S, Mehta JB, Kamath P. Naked eye single tube red cell osmotic fragility test for beta thalassemia trait: population survey. Indian J Hematol 1988;6:187-90.  Back to cited text no. 1    
2.Kattamis C, Effremove G, Pootrakul S. Effectiveness of one tube osmotic fragility screening in detecting beta thalassemia trait. J Mod Genet 1981;18:266-70.  Back to cited text no. 2    
3.Bhuta R. Red blood cell osmotic fragility in health and disease. M Sc thesis Bombay University 1978.  Back to cited text no. 3    
4.Kattamls C, Mallias A, Mentaxotou­Mavromatl A, Matsanlotis N. Screening for beta thalassemias. Lancet 1981;385:1169.  Back to cited text no. 4    
5.Modell B, RazzakA, Hindley N. Thalassemia In the Maldives. Lancet 1990;335:1169-70.  Back to cited text no. 5    
6.Verawala NY, Old JM, Sarkar R, Venkatesan R, Weatherall DJ. The spectrum of beta thalassemia mutations on the Indian subcontinent: the basis for prenatal diagnosis. Brit J Hematol 1991; 78:242-7.  Back to cited text no. 6    
7.Mehta BC, Agarwal MB, Kurlekar N, Varandani DG. Diagnostic criteria of beta thalassemia trait: a study of 171 parents of patients with Cooley's anemia. J Assn Phys India 1982;30:97-98.  Back to cited text no. 7    
8.Galanello R, DeVirgllils S, Addis M, Paglletti E, Ruggeri R, Cao A. J Clin Pathol 1980;33:946-8  Back to cited text no. 8    
9.Mehta BC, Sukumaran PK, Patel JC. Hypochromic anemia caused by beta thalassemia trait. J Indian Med Assn 1970;55:53-4.  Back to cited text no. 9    
10.Mentzer WC. Differentiation of iron deficiency from thalassemia trait, Lancet 1973;1:882.  Back to cited text no. 10    
11.England JM, Fraser PM. Differentiation of iron deficiency from thalasse mia trait by routine blood count. Lancet 1973;1:449-51.  Back to cited text no. 11  [PUBMED]  


  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]

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