Protocol:Red blood cell morphology

Principle
Morphological changes of red cells can be detected in most thalassemia carriers. An examination of a stained peripheral blood smear may be helpful in the evaluation of cases.

Preparation of smears
EDTA anticoagulated venous whole blood or capillary blood is used to prepare a blood film. After drying at room temperature the blood film can be stained with May-Gr&uuml;nwald (or Wright stain).

Reagents

 * 1) May-Gr&uuml;nwald’s stain powder (0.3 g)
 * 2) Methyl alcohol (100 ml)
 * The staining solution is made up by swirling to mix, then warming in a 56 oC water bath for 15 min. Allow to cool to room temperature with occasional shaking, then filter after standing for 24hrs.
 * 1) Giemsa’s stain powder (1 g)
 * 2) Glycerol (66 ml)
 * 3) Methyl alcohol (66 ml)
 * Staining solution is made up by adding the stain to the glycerol, then warming in a 56 oC water bath for 90-120 min. Allow to cool to room temperature with occasional shaking, then filter after standing for 24hrs.
 * 1) Phosphate buffer 0.066 M pH 6.8 (Sorensen’s):
 * KH2PO4: 9.1 g
 * Na2HPO4: 9.5 g (or Na2HPO4.2H2O: 14.2 g)
 * Dissolve in 1 litre distilled H2O to make.066 M buffer of pH 6.8.
 * Add 50 ml of phosphate buffer 0.066 M pH 6.8 to each litre of water used to dilute stains and wash films.
 * Ready staining solutions are commercially available.

Method

 * 1) Smear small drop of blood on slide using a spreader slide at an angle of 45o
 * 2) After drying in air, fix film in a jar of methanol for 15 min.
 * 3) Immerse in a jar of May-Gr&uuml;nwald’s stain freshly diluted with an equal part of buffered distilled water for 5 min.
 * 4) Transfer to a jar of Giemsa’s stain freshly diluted with nine parts of buffered distilled water for 15 min.
 * 5) Transfer to a jar of buffered distilled water, wash with 3 to 4 changes of buffered water and let stand for 5–12 min to differentiate.
 * 6) Stand slide upright to dry
 * 7) After drying examine by microscope under immersion oil.

Interpretation
Red cell morphology is illustrated in Fig. 2.1. Microcytosis, hypochromia and anisopoikilocytosis (variation in the size and shape of the red cells) are the most typical changes in thalassemia. Other less common findings are basophilic stippling and the presence of some target cells. A high percentage of target cells are found in HbC syndromes. Nucleated red blood cells are indicative of bone marrow hyperactivity and can be found in homozygous &beta;-thalassaemia. Polychromasia (blue-grey and slightly bigger red cells) is associated with the presence of reticulocytosis. Howell-Jolly bodies (fragments of nuclear DNA) can be found after splenectomy or in the functional asplenic condition in sickle-cell syndromes, where sickle shaped cells are sometimes seen on the stained film as well.