LABORATORY DIAGNOSIS OF HAEMOGLOBINOPATHIES

LABORATORY DIAGNOSIS OF HAEMOGLOBINOPATHIES

The 1978 International Committee for Standardization in Hematology expert panel on abnormal Hbs has prepared recommendations for laboratory investigation of these conditions.

1.      A complete blood count (CBC)

2.      Electrophoresis at pH 9.2

3.      Test for solubility and sickling

4.      Quantification of HbA₂ and HbF were recommended.

If abnormal Hb was found in these further test including electrophoresis at pH 6.2, globin chain separation and isoelectric focusing were recommended. If the presence of an unstable Hb or Hb with altered oxygen affinity was suspected then heat and isopropanol stability test were recommended. In addition to these test the iron status of patient should be known either by measuring ferritin or by iron/TIBC.

Complete Blood Count:

Thalassemia – Decreased Hb, low MCV <76 fL (normal 90-100 fL), low MCH <27 pg (normal 26-35 pg), High RBC count or count in URL (in IDA RBC count is low and Hb is low), in iron repleted patient suggest diagnosis of thalassemia. These low MCV, low MCH, low Hb, high RBC are the thalassemic indices.

Electrophoresis: 

Electrophoresis under alkaline pH is the common initial screening method for detection and preliminary identification of hemoglobinopatheis. It is done in cellulose acetate or agarose in Tris EDTA borate buffer or barbital buffer in pH 9.2. Visualization of electrophoretogram is done by protein stain like Amido Black or Ponceau S. Electrophoresis at pH 6.0 using citrate buffer is preformed when an abnormal band is noted on alkaline Hb electrophoresis. In acid electrophoresis the overlapping band seen in alkaline pH are further resolved and appear at different position.

Other advanced procedure are IEF, Capillary electrophoresis, HPLC, ESI-MS, DNA analysis, ASO-PCR, etc.

Specific test:

Determining HbH: 

HbH is insoluble tetramer of β₄ and arises in α-thalassemia. If these tetramers are oxidized precipitation occurs that can be viewed microscopically. In laboratory oxidation is achieved by staining unfixed cells with methylene blue or brilliant cresyl blue at 37⁰C. Golf ball like inclusions can be seen in red cells. In α-thalassemia minor 1 cell in 1000 to 10,000 may contain inclusions.

Sickling test: 

This is useful in confirming the presence of HbS in sample following initial electrophoresis at alkaline pH. When fully oxygenated HbS is fully soluble but when deoxygenated, polymerization occurs, forming insoluble fibers that deform red cells giving rigid sickle shape. In lab, deoxygenation and lysis of the RBC is achieved by use of solution of sodium metabisulfite in a phosphate buffer. For this few drops of sodium metabisulfite is added to a drop of anticoagulated blood on a slide. Seal between the slide and cover glass with petroleum jelly/paraffin wax mixture, or with nail varnish. Sickling takes place almost immediately in sickle cell anaemia and should be obvious in sickle cell trait within 1h. After this the slide is observed in microscope to see the sickling phenomena. 

Solubility testing: 

Solubility testing is based on the reduced solubility of deoxy HbS in presence of reducing agent, e.g. sodium dithionite. Here reagent containing sodium dithionite is added to packed red cell and turbidity compared with positive and negative control. In positive, the sample is centrifuged. A positive test will show a dark red band at the top while the solution below will be pink or colorless. Positive test indicates HbS and help in differential diagnosis of Hbs D and G which migrate with HbS.

Test for Unstable Hemoglobins: 

These tests use either heat or isopropanol to precipitate the unstable Hb and must be performed on fresh blood. There are more than 100 unstable Hbs resulting mainly from the interchange of nonpolar amino acid for polar amino acid residue in either α or β chain associated with the heme cleft.

Isopropanol stability test: 

Non polar isopropanol weakens the internal bonds within Hb decreasing the stability of Hb molecules. Normal HbA precipitates within 40 minutes at 37⁰C in presence of 17% isopropanol in pH 7.4 TRIS buffer. Unstable Hbs usually precipitates and gives turbidity within 5-20 minutes under these conditions.

Heat stability test:

  

Positive, negative and test samples are heated to 50⁰C for 2 hr. Normal Hb is stable when heated to 50⁰C. However unstable Hbs precipitates and flocculates to varying extent when similarly treated.

Globin chain analysis: 

Here the globin chains and heme in red cell lysate are first dissociated using urea and dithiothreitol. The dissociated globin chains are then separated electrophoretically at both an alkaline and acid pH.