Protocol:Reticulocyte count and Hb H inclusions

Principle
In thalassaemia carrier screening reticulocyte count does not have a diagnostic value. However in the detection of &alpha;-thalassaemia, especially Hb H disease, the brilliant cresyl blue stain will detect the characteristic Hb H inclusion bodies.

Supravital stains (brilliant cresyl blue or new-methylene blue) are able to stain residual mRNA in immature red blood cells. There are now several automated electronic cell counters able to perform a reticulocyte count using specific RNA staining.

Sample
Capillary or EDTA anticoagulated blood

Reagents

 * 1) Brilliant cresyl blue (BCB)
 * 2) 3% trisodium citrate (3 g in 100 ml of distilled water)
 * 3) 0.9% NaCl

Staining solution is made up using: 1 g BCB, 20 ml 3% Tri-sodium citrate and 80 ml 0.9% NaCl

Dissolve by stirring for 20 minutes, then filter.

Method

 * 1) Add in a tube containing 2-3 drops of staining solution, 2 or 3 drops of blood.
 * 2) Mix and leave at 37 oC for 15-20 min.
 * 3) Make smears, and after drying examine by microscope under immersion oil (Fig. 2.2).

Reticulocytes (red cells containing purple inclusions or filaments) as a percentage of a total of 500-1000 red cells should be counted.

The absolute number is derived as follows:


 * % reticulocytes x RBC number/l
 * 100
 * 100

Normal values:

 * % Reticulocytes = 0.0-2.0%
 * Absolute number = 20-100 x 109 / l

Interpretation of the results
Reticulocyte number is useful in the evaluation of erythropoiesis and is significantly increased when haemolysis (or bone marrow regeneration) is present.

Principle
Under appropriate staining conditions intra-erythrocytic inclusions can be visualized. These inclusion bodies (Heinz bodies) are intracellular haemoglobin precipitates detected by supravital stains. These may be found in some forms of thalassaemia (mainly &alpha;-thalassaemia) and in unstable haemoglobin disorders.

Reagents and method

 * 1) BCB staining as described for reticulocytes.
 * 2) Methylviolet stain (0.5 g in 100 ml of 0.9% saline) can be also used.
 * 3) Dry blood films are made after 30 min, 1h and 3h and examined by microscope under immersion oil.

Interpretation
Heinz bodies appear as simple or more commonly multiple inclusions (Fig.2.3).

They are found in variable percentages (5-50%) in Hb H disease, in unstable haemoglobins and sometimes in very low number (1:1000-10.000 RBC) in -&alpha;/-&alpha; and - -/&alpha;&alpha; carriers.