LABORATORY TESTS USED TO DIAGNOSE

THALASSEMIA MAJOR

A.N. FATHIMA RIFNA

Reg. No. 618185683

Introduction

Thalassaemia is the name for a group of inherited conditions characterized by defective

haemoglobin production. It occurs as a result of gene mutation/deletion and it can be
classified in to α and β Thalassaemia.

Thalassemia results from quantitative reductions in globin chain synthesis. Those with
diminished β globin chains are termed β thalassaemia, whereas those with decreased α chain
production are called α thalassaemia. Severity of clinical manifestations in these disorders
relates to the amount of globin chain produced and the stability of residual chains present in
excess.

The laboratory diagnosis of thalassaemia major starts with,

1. Initial haematological test recommended - Complete Blood Count (CBC)

There are various parameters to be analysed in CBC in diagnosing of Thalassaemia
major.
 Haemoglobin – Low, usually 3 - 9 g/dl
 Red cell indices are critical to diagnosis – Hypochromic microcytic anaemia
 MCV (mean corpuscular volume) is low (usually < 67 fl)
 Mean Corpuscular Haemoglobin (MCH) – Low
 Mean Corpuscular Haemoglobin Concentration (MCHC) –Normal to slightly
decreased.
 RDW (red cell distribution width) is increased.
 Increased RBC count – one distinguishing factor between thalassemias and
other microcytic anemia
 WBC count - increased

2. Peripheral blood film shows,

 Erythrocytes show marked anisocytosis & poikilocytosis
 Predominantly microcytes
 Occasional spherocytes
 Tear drop cells – often seen

2
  Prominent target cells
 Elliptocytes
 Polychromacia – is a feature of immature anucleated erythrocytes
 Basophilic stippling – indicates impaired haemoglobin synthesis, due to the
instability of RNA in the young cell.
 NRBC – Nucleated red blood cells can be used as a marker of erythropoietic
stress and help optimize transfusion therapy in patients with beta thalassaemia
major.

.
Blood film shows target cells, NRBC, microcytosis, poikilocytosis

3. Reticulocyte count

Reticulocytes are young and immature red blood cells, which are passed in to
blood circulation from the bone marrow. They contain remnants of ribosomal
ribonucleic acid in their cytoplasm. These remnants are basophilic and have the
property of reacting with certain dyes, as new methylene blue (supra vital stain)
and form a blue precipitate of granules.

3
 The reticulocyte count used as an indicator of the erythropoietic activity of bone
marrow. Usually normal person contain 0.5 – 2.5 % and infants contain 2 – 5 %
of reticulocytes in peripheral blood.

In thalassaemia major, reticulocyte count is increased, degree of elevation

depends upon severity of thalassaemia.

4. Routine chemistry
 Serum indirect bilirubin – in thalassaemia major, serum indirect bilirubin level
is raised as a result of increased extravascular haemolysis.
 Haptoglobin and haemopexin depleted
 Iron stain – positive iron stain (Perl’s stain) is shown to exclude iron
deficiency anaemia

5. Osmotic fragility test – screening test for beta thalassaemia

The osmotic fragility test is used in determine the resistance of erythrocyte to
haemolysis in decreasing strengths of hypotonic saline solutions. It is very useful
to diagnose of hereditary spherocytosis and screening for thalassaemia. The red
cells that are spherocytic for whatever the cause takes little water in a hypotonic
solution before rupturing than normal red blood cells.

In thalassaemia screening, the osmotic fragility curve shifts to the right due to red
cells fail to maintain the shape.

6. Brilliant cresyl blue stain – (Hb H/ golf ball) – to diagnose alpha thalassaemia Hb H
disease.

When red cell containing Hb-H are incubated with a solution of redox dye
(Brilliant cresyl blue), Hb-H, which is relatively unstable, precipitates and the red
cells are pitted by numerous inclusions.

Hb-H inclusions are appeared as golf balls. Inclusions smaller than Heinz bodies
and are evenly distributed throughout cells.

4
 Red cell contains precipitated Hb-H, ‘golf ball’ appearance

7. Acid elution test (Kleihauer–Betke) for the detection of Fetal haemoglobin (Hb F)

Haemoglobin F (HbF, α2γ2) account for up to 90% of the circulating haemoglobin

at birth. Its synthesis starts to decline during the third trimester, and over the first
year of life it is gradually replaced by adult haemoglobin, HbA (α2β2). Normal
adults have less than 1% of HbF.

In thalassaemia major Hb F is increased significantly and it can be assessed using

acid elution test. The acid elution test is employed to assess the distribution of
hemoglobin F in the red blood cell.

In this test the cell contain Hb F is identified by the fact that they resist acid
elution to a greater extent than normal cells. Therefore, the cells with Hb F appear
as darkly stained (pink in colour) cells amongst a background of palely stained
ghost cells.

5
 Red cells contain Hb F stains pink in colour and adult ghost cells stain pale pink.

8. Alkaline Denaturation Test

To measure the percentage of Hb F in a mixture of haemoglobin, sodium
hydroxide is added to a lysate and, after s set time, denaturation is stopped by
adding saturated ammonium sulphate. The ammonium sulphate lowers the pH and
precipitates the denatured haemoglobin. After filtration, the quantity of
undenatured (unprecipitated) haemoglobin is measured. The proportion of alkali-
resistant (fetal) haemoglobin is then calculated as a percentage of the total amount
of haemoglobin present.

In beta thalassaemia major amount of Hb F elevated.

9. Hb electrophoresis

Hemoglobin electrophoresis is used to evaluate for and identify variant and

abnormal haemoglobins. Alkaline and/or citrate agar electrophoresis is the
commonly used method. Separation of haemoglobins is based on variable rates of
migration of charged hemoglobin molecules in an electrical field.

6
Haemoglobin electrophoresis at pH 8.4 – 8.6 using cellulose acetate membrane is
simple, reliable and rapid.

At alkaline pH, haemoglobin is a negatively charged protein and when subjected

electrophoresis will migrate towards the anode (+). Structural variants, which have
a change in the charge on the surface of the molecule at alkaline pH, will separate
from HbA.

 Hemolysate is prepared from blood in EDTA, or citrate, or heparin.

 The hemolysate is run for electrophoresis where Hb is separated in different
bands.
 This is a migration of charged solutes or particle in liquid medium under the
influence of the electric field.
 In the electromagnetic field, the hemoglobin moves with different rate and
form various bands.
 The patient sample is compared to the normal Hb pattern.
 Each band is quantitated as a percentage of total hemoglobin.

 Hb A1 is the major hemoglobin in the normal RBC.

 While Hb A2 is the minor component (2 - 3 %).

 Hb F is the main hemoglobin in the fetal RBC. There is a very small amount
in the normal adult. ( < 1% )
 In thalassaemia major, Hb F is markedly increased > 90%

7
The pattern of Hb electrophoresis in Thalassemia:

Hemoglobin Normal Thalassemia Major Thalassemia Minor

Hb F < 1% 10 – 90 % -----> β thal + 1–5%

98% -----> β thal 0

Hb A 97% 10 – 90 % -----> β thal + 90 – 95 %

0 -----> β thal 0

Hb A2 1 to 3% 1.5 - 7 % -----> β thal + 3.5 – 7 %

1.5 – 4 % -----> β thal 0

8
10. Hb quantitation

Method allowing the separation of numerous normal and abnormal human

hemoglobin types.

 Method used is HPLC (High Performance Liquid Chromatography)

 Can quantify the haemoglobin accurately
 Hb gel/HPLC migration patterns – Not helpful for α Thalassemia, unless β4
(Hb H) and γ4 (Hb Barts) are present.
 HPLC: Elevated Hb F and HB A2.

11. Molecular Analysis (Globin chain testing, DNA analysis )

 Alpha thalassemia – Multiplex Ligation dependent Probe Amplification
(MLPA) and multiplex PCR – Alpha globin sequencing
 Beta thalassemia – Beta globin sequencing, The test examines the complete
beta globin coding sequence
 Genetic analysis – MLPA: will identify all deletions and duplications

9
References

1. Lecture note – ‘Thalassaemia’ by Lecturer (OUSL)

2. Practical Haematology by Dacie and Lewis
3. https://www.ncbi.nlm.nih.gov/pubmed/27183541

10