LABORATORY ASPECTS OF THALASSAEMIAS
Dr. Mrs. Trupti S. Dankhade (Bobade) *
Consulting Pathologist & Microbiologist "Hi-Tech" Pathology Laboratory, Daffrin Hospital Road, Shrikrishna Peth, Amravati.Ph.:2661396 *
The thalassaemias are a heterogenous group of disorders with genetically determined reduction in the rate of synthesis of one or more types of normal haemoglobin polypeptide chain. This results in a decrease in the amount of haemoglobin involving the affected chain.

CLASSIFICATION

The Alpha & Beta chain of haemoglobin are synthesized independently under separate genetic control. There are two main groups of thalassemia:


  1. One affecting the synthesis of Alpha - chain known as Alpha - thalassemia and

  2. Other affecting the synthesis of Beta - chain known as Beta - thalassemia



Beta-Thalassaemia:

In Beta-thalassemia, there is decreased rate of synthesis of Beta - chain and hence decrease in the amount of the normal Hb-A in red cells.

Clinically, there are two types of Beta-thalassemia:

  1. Beta-thalassaemia major or Cooley's anemia - where there is total suppression of Beta - chain synthesis and is homozygous.

  2. Beta-thalassaemia minor is sometimes asymptomatic condition and is heterozygous.


LAB DIAGNOSIS OF THALASSEMIA:

Firstly, following routine haematological tests are essential:

  1. Haemoglobin level is usually on lower side.

  2. Red cell indices - MCV, MCHC & MCH is significantly reduced.

  3. Reticulocytes count is raised . (Some times even 10% or more)

  4. The white cell count it is usually raised.

  5. There can be the shift to the left of neutrophils.

  6. Platelet count: It is usually normal.

  7. Peripheral Smear Examination:

    It shows microcytic hypochromic picture. Erythrocytes shows marked anisocytosis and poikilocytosis. Target cells are prominent. Polychromasia and punctuate Basophilia is usually present.



Haemoglobin Electrophoresis:
Precise measurement of Hb-A 2 level is required. Hb electrophoresis on cellulose acetate agar at PH 8.6 is widely used.

Electrophoresis Phenotype of Beta-thalassemia
DisorderHaemoglobin (%)
Hb-AHb-A 2Hb-F
B-thalassaemia minor90 - 953.5 - 7.01 - 5
B-thalassaemia major10 - 901.5 - 4.010 - 90


The Alkali denaturation test and acid elution test are used for the measurement of Hb-F.

Bone Marrow Aspiration
It shows hyperplastic erythropoiesis. There is presence of micronormoblast. Methyl violet positive inclusion bodies may be seen.

PREVENTION

Screening programmes to detect asymptomatic, previously unknown thalassaemic heterozygotes and to provide information and genetic counseling have been established.

  • The measurement of red cell MCV using an electronic cell counter is used as basic screening test.

  • Nestroft Test is used to screen the suspected patients of thalassaemia. It is very easy, simple and cost effective test for screening at large scale.



Measurement of Globin Chain Synthesis:


A small sample of fetal blood is obtained at 18th - 20th week of gestation by placental needling under ultrasound guidance or by fetoscopic umbilical vein aspiration. Globin chain synthesis rate in fetal reticulocytes are measured.

Analysis of fetal DNA

Amniotic fluid is obtained by transabdominal amniocentesis during 15th - 18th week of gestation. Analysis of fetal DNA is performed by gene mapping techniques after extraction from fetal fibroblast or chorion. The DNA is cut at specific sequences into small fragments with restriction enzymes obtained from bacteria. The resulting fragments are separated according to size by agarose gel electrophoresis and transferred onto nitrocellulose paper by a process called Southern blotting. The particular gene of interest is identified by hybridization with a specific radioactive probe, followed by autoradiography. The restriction fragments that are complementary to the probe hybridize with it and form a band on the X-ray film.

Direct Identification of mutant genes:

Those disorders in which basic genetic abnormality is either a substantial gene deletion or a base substitution, that alter a restriction enzyme cleavage site, can be identified directly by gene mapping.

ALPHA THALASSEMIA:

In this disorder, there is defective synthesis of Alpha - chain, resulting in decrease of production of Hb that contain Alpha - chain i.e., Hb-A, Hb-A2, Hb-F. Deficiency of Alpha - chain leads to excess of gamma chain in fetus and of Beta - chain in adult.

CLASSIFICATION:
Alpha thalassemia-1:
It shows variable mild anemia and red cell hypochromia. In this condition Hb pattern is normal and Hb - H inclusions are seen usually. The haemoglobin level is normal or only mildly reduced, but the red cells are usually mildly hypochromic and microcytic, and the MCV and MCH are reduced. Hb-A2 is reduced in some patients.

Alpha thalassemia-2:


Shows no abnormality on routine hematological examination. In this, Hb pattern is normal in adults. In new borns, the affected infants may have 1 to 2% of Hb - Bart which they gradually lose over the ensuing months.

Haemoglobin - H disease


Interaction of the Alpha + and the Alpha 0 determinants gives rise to this form of Alpha - thalassemia.

Blood picture shows sever hypochromia, microcytosis, target cells, basophilic stippling and nucleated RBC. There are numerous Hb-H inclusion. The Hb pattern shows 2-40% of Hb-H and remainder can be Hb-A, HbA2, Hb-H.

Haemoglobin-Bart's (Hydrops fetalis)


It is a severe manifestation of Alpha - thalassemia. There is total suppression of Alpha - chain synthesis with gross excess of Gamma - chain. This Gamma - chain tetramer is Hb-Barts. Clinical picture is similar to that of severe Rh haemolytic disease. The blood film shows anisopoikilocytosis, hypochromia, target cells, basophilic stippling, polychromasia and large number of nucleated red cells. Reticulocyte count is high and serum bilirubin is elevated.

Hb pattern consist of 80-90% of Hb-Bart's with a small amount of Hb-H.

Thus, Laboratory aspects comprises a very important role in diagnosis, classification, treatment and prevention of genetically inherited hematological disorder like thalassemia.
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