Leishman stain —Junior Lab Assistant PSC

 PREPARATION OF BLOOD FILMS ON SLIDES

Blood films should be made on clean glass slides. Films made on coverglasses have negligible advantages and are unsuitable for modern laboratory practice. Films may be spread by hand or by means of an automated slide spreader, the latter being either a stand-alone instrument or a component of an automated blood cell counter.

Manual method

Blood films can be prepared from fresh blood with no anticoagulant added or from ethylenediaminetetra-acetic acid (EDTA)-anticoagulated blood. Heparinised blood should not generally be used because its staining charac- teristics differ from those of EDTA-anticoagulated blood. Good films can be made in the following manner using clean slides, if necessary wiped free from dust immedi- ately before use. Slides should measure 75 × 25mm and be approximately 1mm thick; ideally, they should be frosted at one end to facilitate labelling, but these are more expensive.

First, make a spreader from a glass slide that has a smooth end. Using a glass cutter, break off one corner of the slide, leaving a width of about 18 mm as the spreader. A spreader can be used repeatedly unless the edge becomes chipped, but it must be thoroughly cleaned and dried between films.

Place a small drop of blood in the centre line of a slide about 1 cm from one end. Then, without delay, place a spreader in front of the drop at an angle of about 30 de- grees to the slide and move it back to make contact with the drop. The drop should spread quickly along the line of contact. With a steady movement of the hand, spread the drop of blood along the slide. The spreader must not be lifted off until the last trace of blood has been spread out; with a correctly sized drop, the film should be about 3 cm in length. It is important that the film of blood finishes at least 1 cm before the end of the slide (Fig.).

Blood films made on slides. (A) A well-made film. (B) An irregular patchy film on a dusty slide. (C) A film that is too thick. (D) A film that has been spread with inconsistent pressure and using an irregularly edged spreader, resulting in long tails. (E) A film made on a very greasy slide.

The thickness of the film can be regulated by varying the pressure and speed of spreading and by changing the angle at which the spreader is held. With anaemic blood, the correct thickness is achieved by using a wider angle, and, conversely, with polycythaemic blood, the angle should be narrower.

The ideal thickness is such that on microscopy there is some overlap of red cells throughout much of the film’s length (see p. 25). The leucocytes should be easily recog- nisable throughout most of the film. With poorly made films the leucocytes will be unevenly distributed, with monocytes and other large leucocytes being pushed to the end and the sides of the film. An irregular streaky film will occur if the slide is greasy, and dust on the surface will cause patchy spots

The films should be allowed to dry in the air. In humid conditions the films may be exposed to a current of warm air (e.g. from a hairdryer), but this should be in a microbio- logical safety hood.

Labelling blood films

The film should be labelled immediately after being spread. Write in pencil either a laboratory reference number or the name of the patient and the date on the frosted end of the slide or on the film itself (writing on the thickest part, which is least suitable for microscopic examination). A label written in pencil will not be removed by staining. A paper label should be affixed to the slide later. If blood films are to be stored for future reference, apply the paper label in such a manner that it is easily read when the slides are filed.

In a computerised laboratory, bar-coded specimen identification labels are convenient and preferable. These should have the patient’s name, the date and the labora- tory number as well as the bar code.

Fixing blood films

To preserve the morphology of the cells, films must be fixed. This must be done with- out delay, and the films should never be left unfixed for more than a few hours. If films are sent to the laboratory by post, it is essential that, when possible, they are thor- oughly dried and fixed before dispatch.

As you already know, there are various types of Blood cells present in our body, having different shapes and sizes, which take up the stains as per their structures. 

Some of the Components of the blood cells are Basophilic i.e. they have a great affinity for acidic dyes whereas some of the components are Acidophilic i.e. they have a high affinity for Basic Dyes and then there are some components of the cells which are neutral and has the high affinity for neutral stains. 

Therefore, the stains used in the Hematology laboratory are the combination of these three types of stains. Besides the dyes, a buffer is added to the stain which acts as the mordant and enhances the staining reaction, resulting in the better morphology of the blood cells under the microscope.

Romanowsky stains are such types of stains that are universally employed for the staining of blood cells. Almost all the Romanowsky group of stains has two essential components i.e. Methylene blue & Eosin or Azure dye. Methylene blue is a basic dye that has a high affinity for the acidic components of the cell i.e. Nucleus and Eosin/ Azure is the Acidic dye which has the high affinity for the basic components of the cells i.e. the cytoplasm and Granules in some cells.

Most of the Romanowsky stains are prepared with Methyl alcohol (Methanol) so that they act as a fixative as well as the cellular stain. There are 4 different types of Romanowsky stains commonly used in Hematology laboratory for staining the blood cells –

• Leishman Stain

• Giemsa Stain

• Field’s Stain

• Wright’s Stain

Leishman Stain

A brief Description of Leishman Stain….

Leishman stain is a mixture of Methylene blue, and Eosin dye, prepared in Alcohol medium and diluted with buffer or distilled water during staining procedure. Leishman stain is a differential stain that is used to variably stain the various components of the cells and it can be used to study the adherence of pathogenic bacteria to the human cells. It differentially stains the human and bacterial cells and appeared as purple and pink colored bodies respectively. The Leishman stain is one of the best stains for routine blood stain to stain the Peripheral blood smear for the examinations of blood film under the microscope and is satisfactory for malaria and other blood parasites. Giemsa stain gives better results in parasitic studies.

Principle of Leishman staining

Leishman Stain is a neutral stain for blood smears which was devised by the British surgeon W. B. Leishman (1865–1926). It consists of a mixture of eosin (an acidic stain), and Methylene blue (a basic stain) in Methyl alcohol and is usually diluted and buffered during the staining procedure. It stains the different components of blood in a range of shades between red and blue.

It is based on a methanolic mixture of “polychromed” Methylene blue and eosin. The methanolic stock solution is stable and also serves the purpose of directly fixing the smear eliminating a prefixing step.

Leishman stain is commonly used when there is need to examine the Blood smear for the Various blood cells, Differential Leucocyte count, Type of Anemia, Toxic Granules & Platelet count etc. and also used to differentiate nuclear and cytoplasmic morphology of the various cells of the blood like Platelets, RBCs, WBCs as well as for the parasites. This stain is the most dependable stain for Peripheral blood film examination.

The working principle of the Leishman stain is same as described above. As it is a type of Romanowsky stains, it contains both the Acidic and Basic dyes which have the affinity for Basic and Acidic components of the Blood cells respectively. The acidic dye, Eosin, variably stains the Basic components of the cells i.e. the cytoplasm, Granules etc. and the Basic dye, Methylene blue stains the Acidic components, especially the Nucleus of the cell. The stain must be diluted for use with Phosphate buffer to pH 6.8 or 7.2, depending on the specific technique used. The pH 6.8 is preferred when the morphology of blood cells is to be examined and pH 7.2 is good for parasitic studies.

Procedure of Preparing Leishman stain in Laboratory

Weigh out 0.2g of the powdered dye, and transfer it to a conical flask of 200–250-ml capacity. Add 100 ml of methanol and warm the mixture to 50°C for 15 min, oc- casionally shaking it. Allow the flask to cool and filter the solution. It is then ready for use, but it will improve on standing.

Nowadays, commercially prepared Leishman stain solution is used in most of the laboratories which are then diluted in various ratios for different purposes.

STAINING METHODS

• Air-dry the film and flood the slide with the stain. 

• After 2 min, add double the volume of water and stain the film for 5–7 min. 

• Then wash it in a stream of buffered water until it has acquired a pinkish tinge (up to 2min). 

• After the back of the slide has been wiped clean, set it upright to dry.

The color of Nuclei by Leishman Stain

Chromatin    Purple

Nucleoli    Light blue

The color of Cytoplasm by Leishman Stain

Erythrocytes    Pink

Reticulocytes    Dark blue

Lymphocytes    Blue

Monocytes    Grey blue

Neutrophils    Bluish Pink

Basophils    Blue

The color of Granules by Leishman Stain

Basophil    Purple black

Eosinophil    Red orange

Neutrophil    Purple

Platelet    Purple