NESTROFT

NESTROFT is an acronym for Naked Eye Single Tube Red Cell Osmotic Fragility Test. As the name suggests, NESTROFT is used to assess the osmotic fragility of red cells.

2 ml of 0.36% buffered saline solution is taken in one tube and 2 ml of distilled water is taken in another tube. A drop of blood is added to both tubes, which are left undisturbed for half an hour at room temperature. The contents of both tubes are then shaken and held against a sheet of white paper on which a thin black line is drawn. The line is clearly visible through the contents of the tube with distilled water. If it is not visible through the tube with buffered saline, the test is considered positive for Thalassaemia Minor.

For a further confirmation, the tubes are left undisturbed for a few hours. A positive test for Thalassaemia Minor will show a red sediment at the bottom of the tube, whereas the one with distilled water will be uniformly pink.

A positive test is also indicated in the case of iron deficiency anaemia, liver disease and other haemoglobinopathies. Therefore this test is indicative but not conclusive for Thalassaemia Minor.

NESTROFT does not need any sensitive equipment and is very inexpensive.

Red Cell Indices

A Complete Blood Count (CBC) for determination of Red Cell Indices is sometimes used as a first-step screening for Thalassaemia Minor. It involves determination of Mean Corpuscular Volume (MCV) and Mean Corpuscular Haemoglobin (MCH). Accuracy of these red cell indices will depend on use of electronic particle counters, which are expensive and need regular maintenance, standardization and quality control.

Reduced levels of MCV and MCH are indicative of Thalassaemia Minor. But these levels are also seen in cases of Iron Deficiency Anaemia (IDA). To differentiate between Thalassaemia Minor and IDA various functions based on Haemoglobin, Red Cell Count and Red Cell Indices have been devised.

However it is necessary to carry further confirmatory tests using other accurate methods on the suspect samples.

Electrophoresis

Haemoglobin (Hb), the oxygen-carrying protein comes in different molecular forms like HbA, HbA2, HbF, HbS, HbC, HbD, HbE, HbM etc. these different Hb types carry different electrical charges.
In Electrophoresis an electrical current is passed through the Hb in the blood sample. The different Hb types separate at different rates in the form of bands. By comparing the bands with the normal pattern, one is able to determine the different Hb types and their quantities.

HbF (Foetal Haemoglobin) is normally present in newborn babies. Very soon this starts depleting and Adult Haemoglobin (HbA) starts getting produced. In a normal adult the following ranges can be seen: HbA: 95-98%; HbA2: 2-3%; HbF < 2%. Thalassaemia Minor is indicated when the levels of HbA2 > 3.5 % and HbF > 2 %

High Performance Liquid Chromatography(HPLC)

The most accepted method of choice for screening for Thalassaemia Minor and other Hb variants is the Cation-exchange HPLC. As discussed earlier, the different forms of Hb carry different electrical charges.

The cation exchange HPLS carries a negative-charged solid support to which all the positive charged molecules get attracted. The adsorption to the solid support depends on the number and location of the charge on the Hb molecule. The molecules with the weakest ionic interactions are disrupted first. A dual wavelength photometer monitors the adsorption. The data is processed and presented in the form of a chromatogram. The different peaks are identified, and represent the different Hb molecules.

The HPLC system offers an automated, quantitative, fast and accurate method for determination of HbA2, HbF and other Hb variants, with the facility of a hard copy report.

DNA Analysis

Thalassaemia and other Haemoglobinopathies occur due mutations which cause the gene to become defective. These defective genes have been identified and sequenced. DNA analysis allows us to identify the genotype and clarify most of the diagnostic problems.

Globin Chain Synthesis

Analysis of Globin Chain Synthesis is a very useful method for diagnosis in cases where the type of thalassaemia is not clear. Globin Chain Synthesis analyses the overall output of globin genes, and may reveal hidden globin gene defects like silent Thalassaemia Minors. This method is difficult, time consuming, and calls for expertise in the personnel carrying out the analysis.

Choice of Method of Screeing

Various factors like financial feasibility, level of accuracy, ease of screening, stability over time of the sample etc. come into play for deciding the method of screening to be used. The various methods that could be used for population screening are:

1. NESTROFT followed by Electrophoresis/HPLC.
2. Red Cell Indices followed by Electrophoresis.
3. Red Cell Indices followed by HPLC.
4. HPLC.
5. Red Cell Indices along with HPLC.

The following three methodologies are being advocated for screening for Thalassaemia Minor:

1. An initial screening by looking at the Red Cell Indices. A second screening using HPLC may be carried out on samples having reduced MVC and MCH. This will help in bringing down the cost of population screening, where finance is a constraint.
2. Subjecting all samples to screening using HPLC. This method could be used for screening of a high-risk population, where finance is not a constraint.
3. Carrying out a double screening of Red Cell Indices and HPLC on all samples. This may be necessary in population where there is co-existence of both Alpha and Beta Thalassaemia and other haemoglobinopathies.

Though DNA Analysis and Globin Chain Synthesis can confirm the diagnosis, it is always advised to study the haematology of the parents.